Download Instructor`s Manual

MICROBIOLOGY STUDENT DATA SHEET
The following information about you and your academic background will be used to help us to better
understand your needs. All information will be treated with confidentiality.
Name:______________________________
Lab sec: __________
Semester: _________
Address:_______________________________________________________________________
________________________________________________________________________
Phone #:_____________________________ e-mail address:_______________________________
Student ID#:_________________________
Emergency Contact Person and Phone #:_____________________________________________
_____________________________________________
1. Academic preparation
Check
courses
taken
Classes taken
When
Grade
earned
Comments?
Introductory chemistry
Chem 101
Additional chemistry
Please list
Anatomy (Bio 260)
or A&P first semester (250)
Physiology (Bio 261)
or A&P second semester
(Bio 251)
Introductory biology
(Bio 100, 102)
Core biology
200-level courses
Algebra
(Math 090,095,102)
English
(Eng. 101,102)
Other
(Medical Terminology/
Health/etc.)
2. Have you ever been enrolled in Microbiology before this semester?
Yes ___ No ____
If yes, please explain the circumstances (when, where, why you are repeating)
______________________________________________________________________________________________
______________________________________________________________________________________________
3. What is your current course load? Total number of units_____
List courses:
______________________________________________________________________________________________
______________________________________________________________________________________________
Continued on back
4. Academic skills self assessment:
Excellent Good
Average
Weak
Very Bad
Reading speed
Reading comprehension
Writing skills
(grammar/
spelling)
Verbal
Communication
Math
Problem solving
Note taking skills
Memorization of facts
Comprehension of complex
or abstract ideas
Test taking skills
Stress management
Time management
Ability to work in teams
5. Do you have any difficulties that could effect your ability to function in the course?
Yes
No
Difficulty
Vision
Hearing
Movement
Learning
Medical
Comments: any special assistance the instructor can provide?
(any condition that weakens immune response)
Psychological
Financial
Job
Transportation
Family
Other
6. What is your career goal? (Why are you taking this class)?
______________________________________________________________________________________________
______________________________________________________________________________________________
______________________________________________________________________________________________
Professor’s comments: ________________________________________________________________
____________________________________________________________________________________
____________________________________________________________________________________
Discussed with student: _________________________________________________________________
____________________________________________________________________________________
____________________________________________________________________________________
MICROBIOLOGY LAB SYLLABUS
Lab Instructor:
Contact Information:
Lab Period:
Room HLS 218
1. INTRODUCTIONS: fill out your student data sheets and hand them back in today.
2. ATTENDANCE: Attendance is mandatory. I will call roll each day. Labs are an extremely
important part of this class and cannot be “made-up”. There will be no switching of lab sections
to make-up for missing a lab period. Any missed lab exams might be made up if approved by the
lecture instructor prior to the date of the exam (where possible and granted on a “case-by-case”
basis) and only with documented proof of the reason for the absence (e.g. physicians note).
Lastly, students with 3 consecutive absences will be dropped.
3. REQUIRED MATERIALS:
Lab manual: Benson's Microbiology Application by Alfred E. Brown; Short Version, 12th
Ed
Lab coat or apron - At times, we will be working with potentially pathogenic organisms as
well as using various stains and dyes. Students should have a lab coat to protect themselves
and their clothing. For safety reasons, all students must wear closed toe shoes and long pants.
Individuals with long hair will be required to pull their hair back. Gloves will be provided
and must be worn when working with microorganisms.
Lab Supplies: colored pencils, black Sharpee permanent marker, 3 ring binder, filler paper,
dividers
4. STUDY TIPS:
a) You will need to spend about 5 hours a week studying for just the lab portion of this class.
b) Make yourself a calendar of the due dates for your report and exam dates.
c) Print handouts as assigned (check lab schedule) from Blackboard postings before coming to
class.
d) For every lab, you must read the lab assignment BEFORE class. During lab, fill out any
questions for the exercise and bring your manual to me to sign off before you go home. I will
check to make sure you have cleaned your lab countertop, cleaned labels off your glassware,
put equipment away properly before I sign off on your lab manual.
5. EXAMS: There are four lab exams worth 75 points each. All exams are from the previous unit
only, nothing cumulative. All exams are questions on paper; no microscopes. Questions are
multiple choice, true/false, matching, and fill-in-the-blank.
6. REPORT: You will write one report on your “unknown” organism, worth 100 points.
7. GRADING POLICY: Note: A passing score for the lab is required in order to pass the
course [i.e. Sixty percent of the lab points (240 points out of 400) must be achieved in order to
pass the class.]
Four 75-point lab exams......................................……….......…300 points
Unknown report……………………………………………….100 points
Total points for lab = 400
8. LAB SAFETY: Rules of Conduct and Clean-up Procedures handout
9. LAB SCHEDULE
10. LAB WORK TO DO TODAY
a) SPECIMEN SIZE DETERMINATION: Handout
b) LAB MANUAL EXERCISE 1: Microscope
Microscope Care and Use handout
c) LETTER “E” Worksheet
Microbiology Laboratory
RULES OF CONDUCT AND CLEAN UP PROCEDURES
The following rules have been established to protect you and your lab neighbors. It is wise and
good technique to treat all microorganisms as potential pathogens and all lab equipment as
potential accidents waiting to happen. Failure to observe the rules jeopardizes your safety and
that of your classmates. Gross negligence will be reprimanded.
Rules of Conduct
1. ATTIRE: Lab coats and closed toe shoes are required and must be worn at all times in lab.
Open-toed shoes are not permitted. Anyone without a lab coat, or wearing open shoes cannot
attend lab that day.
a. Fold lab coats and keep them in their storage nook when not in use.
b. Before removing your lab coat from the lab for laundering or at the end of the
semester, it must be sterilized. Bring a paper grocery bag to place the coat in.
Clearly label the bag with your name and lab section and turn it in to the lab
technician for autoclaving. Speak with the lab technician before leaving your coat to
determine when they will be sterilizing and can include you coat. This will ensure that
you will have time to wash the coat and return it in time for your next lab meeting. Coats
not collected within one week of the close of the semester or upon leaving the class will be
confiscated.
c. Do not wear your coat outside of the lab. If you should need to leave the
lab during class, remove your coat and hang it over the back of your
seat. Upon returning to lab, put the coat back on.
2. SMOKING, EATING AND DRINKING are not allowed in the lab, including water, gum,
and mints. During lab you should not put anything in your mouth i.e. pencils, fingers.
3. PIPETTE with pipette aids only. “In the old days…” it was a common practice to pipette by
mouth. Today it is against lab safety protocol to use anything but various types of pipette aids.
Take care not to draw anything into the pipette aids. If any cultures or reagents do get drawn into
a pipette aid, that pipette aid must be withdrawn from use and cleaned appropriately by the lab
technician before it can be put back into use. This is to prevent contamination of cultures and
personnel.
4. LABEL everything clearly (tubes, petri dishes, slides) with the name of the organism, type of
media or stain, date, your name (no initials), lab period and any other pertinent information that
you may need to identify and retrieve your materials. There will be labs when each student will
have several cultures. To prevent loss of cultures or confusion between them, it is necessary to
label. Label petri dishes on the bottom and along the periphery so as not to obscure examining
the plate. Label tubes along the length of the tube. DO NOT write on the white paint on the test
tubes, as the writing cannot be removed and we reuse our tubes. Do not put tape on tube caps; the
adhesive is not easily removed and makes it difficult for cleaning. When you use a marker or
label on glass flasks and tubes, make sure all labels are removed, and all grease marks are erased
before you leave. Put equipment back where you got it.
5. KEEP LAB COUNTERTOPS CLEAR AND CLEAN. Store packs, purses, etc. out of the
way, under the counter (knee hole). Your lab counter area must be wiped down with disinfectant
when you come in and when you leave. Throw the paper towels into the regular trash
afterwards, NOT in the red biohazard bag. The biohazard bag is for any and all hazardous
materials, including a toothpick you put in your mouth, gloves, disposable Petri dishes, etc. Use
the regular trash for everything else. Before you leave, I need to check that your area has been
disinfected, left clean, and that the microscope has been put away properly.
6. GAS LINES: Make sure the gas lines are completely shut off when not in use. Check again
before leaving the lab for the day. Report any gas leaks to the instructor or the lab technician.
7. WASH YOUR HANDS with soap and water before leaving the lab. Use the sinks in front of
Deck 1 and 3. Each sink has a soap dispenser and paper towel dispenser.
8. TURN OFF all phone/pagers while in lab.
Clean up Procedures
1. Biohazard Containers:
Biohazard containers are designed to receive biohazardous matter. This is largely due to the
lining. which is a special red bag that can withstand autoclaving. All biohazard materials are to
be disposed into these red containers.
a. Remove the bag when it is ¾ full. Tie the bag end into a knot.
b. Take the full bag to the prep room and give it to the lab technician for further
processing.
c. Line the container with a new red bag. New bags are located in drawers in the
front of the room marked with red tape.
2. Test tubes:
a. Remove all markings from test tubes and caps, using kimwipes or cotton balls and
ink remover or alcohol.
b. Boil all culture tubes in a boiling water bath with caps loosened for 30 minutes.
Begin counting time after water begins to boil.
c. Remove caps and dump tube contents, except Durham tubes into a biohazard
container – NOT THE SINK. Durham tubes can be “caught” by pouring the
suspension through a strainer over the biohazard container. A strainer is
located at each sink.
d. Rinse out all traces of media from culture tubes with water and a test tube brush.
Brushes are located at each sink. DO NOT USE SOAP OR CLEANSER. Place
brushes into the containers designated to keep them free of soap that may collect
in the sink from hand and slide washing. These products can leave a residue that
kills our cultures when we reuse the tubes.
e. Place rinsed tubes with caps removed in the wash basin near the prep room door.
A separate container for Durham tubes is inside the washbasin.
3. Plastic petri dishes:
These are placed into the biohazard container when your work with them is completed.
It is not necessary to remove any writing.
4. Pipettes:
Pipettes are placed tip down into special container for pipettes – a pipette bin. The pipette bin
should be lined with a biohazard red bag. Pipette sleeves can be disposed of into the trash unless
you replace a used pipette into the sleeve. In this case the whole pipette and sleeve needs to be
placed into the pipette bin.
5. Micropipette tips:
Micropipette tips are ejected into special containers for them. These will be labeled and placed on
counter tops when experiments require micropipettes.
6. Prepared slides: (price range from $2.25 to $16.00)
a. Do not break slides due to careless handling or improper microscope technique.
b. Remove all traces of oil from each slide. Wipe oil away with a kimwipe. If
necessary, one or two drops of alcohol can be used to remove residual oil. Take
care not to remove permanent slide labels.
c. Dry the slide with kimwipes and return to the proper tray. Trays are labeled on
their ends with the name of the slides.
7. Student prepared heat fixed slides:
These are washed and reused. Each student will have a box of clean slides in their drawers for
their use.
a. Remove immersion oil with kimwipe
b. Wash slides clean with a non-abrasive cleanser; do not use the brush used
for tubes.
c. Rinse thoroughly and dry slide with kimwipes, pass through bunsen burner
flame once and return to your slide storage box.
8. Wet mount preparation:
These are non-heat-fixed slides and include the hanging drop method (depression slide
$2.25).
a. Saturate slide with coverslip in place with disinfectant for 15 minutes; capillary
action will draw the disinfectant under the coverslip.
b. Wash slide and cover slip with non-abrasive cleanser and rinse thoroughly
with tap water. Take care in handling and cleaning coverslips. They break
very easily and can cause cuts.
c. Dry slide with kimwipes, pass through a Bunsen burner flame once and return
to your slide storage box.
9. Contaminated cotton swabs (applicators) should be placed back into their sleeve
immediately after use and then discarded into the biohazard container.
11. Culture spills:
a. Flood the area with disinfectant.
b. Cover with paper towels and let it sit for 15 minutes.
c. Notify the instructor or lab technician.
d. Alert students as to the location of the spill while paper towels are in place so as
to prevent anyone from slipping on it.
e. Wipe up the area with paper towels and place all towels in the biohazard
container, along with your contaminated gloves.
f. Using a hand broom and dustpan, sweep up any glass and dispose of it into a
biohazard container.
12. Broken glass:
a. Notify the instructor or lab technician.
b. Carefully sweep up all broken glass. A hand broom and dustpan are located at
each sink on either end of the lab.
c. Deposit broken glass into boxes and/or plastic container marked “BROKEN
GLASS”. Never put broken glass into the trash cans, as maintenance personnel
could be injured.
d. Broken glass as a result of a culture spills is disposed of as instructed above for
culture spills.
13. IN CASE OF INJURY
a. Notify the instructor or lab technician immediately.
b. In the event of a cut, instruct the injured person to apply pressure to control
bleeding.
c. In the event of a burn, have the injured person flood the burn with cold water.
d. If lab personnel are injured or absent, call the campus health services at X-4494
using your cell phone or the phone in the prep room if you have no cell phone.
In the event of a life threatening emergency call X-1222.
e. For other emergencies or if the health services does not answer, call the
campus police emergency number X-4492.
7. FIRE SAFETY: Fires in this lab are started from flammable items becoming ignited by
Bunsen burners. Do not leave a lit Bunsen burner unattended. Keep alert to the hazards of open
flames and boiling water. Keep flammable materials away from the flame and never reach over
a burning flame. Remember, a lab coat can be taken off and used to put out a fire if someone’s
hair catches fire, etc.
14. FIRE EVENT: Immediately notify staff
a. In the event of fire, use the fire extinguisher located on the wall next to the prep
room window.
b. Should a student’s clothing catch fire, assist student to the shower and pull lever
to release shower water. A fire blanket is also located next to the fire
extinguisher that can be draped over the student to help extinguish the fire.
c. Should a student’s eyes get splattered with chemicals, assist the student to the
eye wash and open wash basin; have student lower face into basin to rinse eyes
for several minutes.
15. At times there may be a need to specify additional procedures for special experiment. These
will be addressed at that time.
Unit One Notes
MICROSCOPE CARE AND USE
Microscopes: (very expensive!!!)
a. Handle with care. They are heavy instruments which give the illusion that they
are not delicate. Wrong! They must be set down gently and therefore it is;
necessary to use TWO hands when moving them around.
b. Clean ALL traces of oil from ALL lenses and stage (and any other place you find
it) at the end of each lab.
c. YOU WILL BE HELD ACCOUNTABLE FOR YOUR ASSIGNED MICROSCOPE
WHETHER YOU USED IT OR NOT ON ANY PARTICULAR DAY. Always
check it before you leave the lab.
The arm should face you as you lift it up. Hold with one hand under base, gently set on desk near
edge, and then turn the arm away from you. Fold up dust cover and put it in the drawer. Here are
some booklets to help you identify the parts of a microscope.
PARTS OF A MICROSCOPE: Find the substage adjustment knob (left side, under stage).
What happens when you turn it? It raises and lowers the condenser. When you are done with the
microscope, leave the condenser in the highest position; this is called racking up the condenser.
Everyone point to the condenser knob. The ocular is the eyepiece. It magnifies ten times and
focuses the image on the retina. There are two oculars. Do not touch your eyelashes to them or
they will get oil on them. To clean, squirt alcohol onto lens paper and wipe, then wipe with dry
lens paper.
The left ocular has a diopter adjustment ring (knurled knob with ridges), with plusses, minuses,
and a zero on it. What happens when you move it? It moves the left ocular up and down. The
purpose of this is to allow the left eye to be focused independently of the right eye. The coarse
adjustment knob is for the right eye focus. Between the oculars is a disc with numbers on it.
This determines the interpupillary distance in mm (distance between your pupils). You can adjust
this so that you can look with both eyes. That will help you see one image instead of two. If you
still see two images, you have a convergence problem and you’ll need to keep one eye closed.
EYE DOMINANCE
We use both of our eyes, but one more than the other. Take a piece of paper, folded into a tube
lengthwise. Hold the tube 12” away from your face and look at someone. Have that person tell
you whether they see your right or left eye in your tube. That is your dominant eye. Do this
now with your partner and have them tell you which eye is dominant on you.
One of your oculars has a pointer, and the other may have a ruler (ocular micrometer) used to
make measurements. When you are using the pointer, switch that ocular to the dominant eye. To
switch the oculars, just pop them out, make sure you don’t drop them. Don’t leave them off long,
or dust will get in. To turn the pointer, just turn the ocular or move the stage so the specimen
meets the pointer. Always use the pointer to show me things when you have a question about
something you see in your microscope.
The revolving nosepiece holds the objectives. Practice turning it; listen and feel for the objective
locking into place. You need to know the following about the four objectives: The scanning
objective (red ring) is the smallest, and magnifies 4x. Since the ocular is 10x, the total
magnification is 40x. The low power objective is 10x, total mag = 100x. The high dry objective
is 40x, total mag = 400x. The oil immersion objective is l00x, total mag = 1000x.
FOCUSING: There are two knobs, one for coarse and one for fine adjustment. Fine adjustment
is smaller knob that is on top of the course adjustment knob and sticks out from it. The coarse
adjustment knob moves the stage a lot, and the fine knob moves it a little. Changing the focus
knobs changes the distance between the stage and the objectives. This distance is called the
“working distance”. Always start with the scanning objective, since it is the only one that can’t
hit the stage and break the lens and the slide. Then make sure the condenser is “racked up”, which
means it is in the highest position. Then “rack up” the coarse knob (turn it so the stage is all the
way up). Look at the slide, then lower the stage with the coarse knob until it comes into focus.
Only after that can you switch to the next power up (yellow low power). If you are not on the low
scanning lens, you may ONLY use the fine adjustment knob. Never use the coarse adjustment
knob if you are not using the red, low-power lens, or you might break the slide and the
microscope lens.
PARFOCAL: This term refers to the factory adjustment which means that once you are
focused with the scanning objective (the red, low-power lens), you will remain focused with
all of the objectives (except for fine adjustment for minor corrections). If you lose sight of
what you are looking for on the slide, always go back to the scanning objective and start
again.
Inside the condenser is a lever: the iris diaphragm lever. This opens and closes like the iris in
your eye (pupil) to regulate the amount of light allowed in. The condenser takes light from
the lamp and makes the rays focus into a small area on the slide. The iris does the following
four things:
a. Regulates light intensity
b. Contrast: (when iris is open, the contrast decreases)
c. Depth of field (when iris is open, only the foreground is in focus. When the iris is
closed, the depth of field increases and everything is in focus.
d. Resolution (sharpness of image). Resolution is best when iris is open all the way.
STORING THE MICROSCOPE: The arm should face your body, the dust cover in place, the
AC (power) cord is stored in the same way you got it. The condenser should be racked up. The
power switch should be off. The voltage regulator should be turned to zero or the lowest setting.
It is located on the side near the base or below the power switch. The stage should be racked
down by using the coarse adjustment knob (next to the arm). The scanning objective should be in
place, not any of the higher power objectives. Clean off the oil and other debris; wipe the ocular
lens with lens paper only.
BE ABLE TO MATCH THE PARTS OF A MICROSCOPE TO A PHOTO OF A
MICROSCOPE.
GETTING TO KNOW YOUR MICROSCOPE
1. What is the working distance?
Since you start with the lowest power, as you increase magnification, working distance decreases.
Scanning objective has the best depth of field and the greatest working distance. When you are
focused with one objective, you are focused with all of them. What is this called? PARFOCAL.
2. What are the functions of the parts of the microscope? Know the four functions of the iris
diaphragm (resolution, contrast, brightness, depth of field). Substage adjustment knob (moves
condenser up and down – when down, there is poor resolution). Diopter ring (allows left eye to
focus independently. Start with stage up, then bring down to focus the right eye.
3. Understand all the parts of the microscope and the associated terms.
4. Why does an image appear upside down and backwards? To answer this, you will look today at
the letter “e” at 40x, 100x, and 400x.
FOCUS SEQUENCE
1. Stage should be racked down.
2. Scanning objective should be in place.
3. Open stage clips (spring loaded).
4. Mount slide onto stage with specimen approximately centered.
5. Move stage up with coarse adjustment knob.
6. Turn power on.
7. Adjust your chair so you are not bending.
8. Increase voltage until you see some light, but stay below maximum.
9. For most slides you need high contrast, so open the iris to the maximum resolution. When
you look at live cells, close the iris to decrease resolution.
10. Adjust oculars for the interpupillary distance until you see just one circle of light called
the FIELD. The type of microscope we have is a Brightfield.
11. Turn coarse adjustment knob to lower the stage slowly while looking through ocular for
the image to appear.
12. Make sure the specimen is still centered.
13. Close your left eye and use the coarse adjustment knob to focus the right eye.
14. Close your right eye and use the diopter right to focus the left eye.
15. Use the revolving nosepiece to move to the 10x objective.
16. Image brightness will now decrease, so turn up the voltage control if needed.
17. Adjust the image contrast with the iris diaphragm lever.
18. Use the fine adjustment knob to focus. DO NOT TOUCH THE COARSE ADJUSTMENT
KNOB AGAIN! If you lose sight of the specimen, go back to the scanning objective.
19. When finished observing at that power, turn revolving nosepiece to 40x. You will see a
decrease in depth of field. If you are looking at a live specimen in water, you will need to
use the fine adjustment knob to follow it around.
20. Adjust for image brightness with voltage.
21. Adjust for contrast with iris.
You can use the x/y adjustment knobs to move the stage around if needed.
When you look at something under a microscope, you must draw a picture of t.
When you draw a picture of something, you must label it with what it is, and what structures it has
with arrows pointing to them. Use this to study. Colored pencils will help.
Take only one slide at a time. When done with a slide, bring it back and get another.
SLIDE TO LOOK AT NOW: Letter “e”
Why did the letter e look upside down and backwards?
The image gets bent as the light rays get bent through the lens, which is biconvex. Light entering
top and bottom of lens gets bent (refraction). This is caused because speed of light is changing. As
they travel through air, light rays go 186,000 miles per second. When they bump into some glass,
they slow down and get bent. Lenses allow us to magnify the image by focusing the light rays at a
point. As we move image back and forth, it allows us to focus. Then it hits mirror and is sent to
oculars and is bent again with your eye lens. Wind up with a focal point. That’s why when you
move slide to left, appears to go right. Our eye lens bends things upside down and backwards;
brain learns to switch the image. No brain in the microscope.
Under hi-dry look at RBC (pink) and WBC (purple nuclei). There are not as many WBCs as
RBCs. Practice putting the pointer on a structure and draw and label a picture of the WBC with
cell membrane, nucleus, and cytoplasm. Label the RBC with just cell membrane and cytoplasm
because there is no nucleus.
CLEANING UP OIL
Remove the oil from the slide before you return it to the slide tray. You only need to clean the oil
off the lens before you leave.
The oil immersion lens has a sealer around it so the oil cannot seep in, but the other objectives are
not sealed, so don’t get oil near them. First, put your finger on the slide label as you lower the
stage or the slide will stick. Remove the slide; take a piece of lens paper torn in half, and use half
to wipe off the oil from the slide and throw the paper in the regular trash. Take the other half of
lens paper, spray it with alcohol, and wipe what’s left of the oil smear off the slide.
Take another piece of lens paper torn in half. Take one half, fold it in half, and just ouch it to the
oil immersion lens. DO NOT RUB OR IT WILL SCRATCH THE LENS ($150). Dab it again at a
clean spot on the paper. Dab again at a clean spot until no more oil is coming off. Take the other
half of lens paper, fold it in half, spray with alcohol, and dab it to the lens as before. Use the
alcohol soaked paper also a little on the blue (high dry) lens to be sure it is clean. Then use it to
clean the stage or condenser
SPECIMEN SIZE DETERMINATION
Exact measurements can be accomplished with the use of micrometers. (see lab exercise #4). In
the absence of this equipment, size determinations can be approximated with a good deal of
accuracy by using the following table and formula.
Micrometers ( microns) = μm
Objective
(numerical
aperature)
Scanning
(0.1)
Low
(0.25)
high dry
(0.65)
oil
immersion
(1.25)
Objective
Ocular
Total
Diameter
Magnification Magnification Magnification of field
view (μm)
4X
10X
40X
5000
10X
10X
100X
2000
40X
10X
400X
500
100X
10X
1000X
200
SIZE = diameter of field X % of diameter that
of view (μ)
the specimen covers
To determine the approximate size of an object in the field of view you must approximate the
amount of space the object takes and multiply by the diameter field of view. eg. specimen length
takes up approximately 30% and the width approximately 15% of the field of view at low power.
length = .33 X 2000 = approximately 660μm (or 2000/3) = 666μm
width = .15 X 2000= approximately 300μm (or 2000/7) = 286μm
Therefore the approximate size of specimen is 666μm X 286μm
2000μm
2000μm
33%=660
μmμmμm
15%=285
Lab 2: Aseptic Technique
Some of the organisms we use in this lab are considered to be Bio Safety Level Two (BS2). BS2
microbes are potentially pathogenic (cause disease) if a person is inoculated with a large dose or if
the person is immuno-compromised. They require autoclaving (steam heat sterilization) and have
to be handled by aseptic technique (we will discuss that later today). BS3 microbes have to be
handled with safety equipment under the hood. BS4 microbes require a space suit type of gear.
This room has negative pressure, so when you open a door, air moves into the room, to prevent
airborne pathogens from escaping. All the air in this room is circulated about every eight minutes.
We need to eliminate contamination within the lab as well. We need to grow and study these
organisms without contaminating anything.
GROWTH MEDIUMS
Nutrients are added to agar (a seaweed product) and heated to a liquid, and sterilized. The sterile
liquid agar can be poured into a Petri dish and cooled. The Petri dish is then referred to as a
“plate.” Sometimes liquid agar is placed in a tube which is tilted 45° while cooling. This is called
a slant. Sometimes the nutrient medium is a broth which does not solidify. Tubes with this
medium are called nutrient broth tubes. All of these types of mediums are made for the purpose of
being inoculated, which means having a tiny amount of a bacterial culture streaked across the
surface. After inoculation, the plate or tube is usually incubated for at least 24 hours to encourage
growth of the sample.
Broths and Slants
Broths are the same nutrients as NA (nutrient agar), but without the agar. They do not
have to be boiled, just stirred. They are placed in tubes instead of plates and autoclaved as
usual.
Slants are made exactly as NA, except they are poured into tubes instead of plates. After
they are removed from the autoclave, they are placed on slant racks to cool so the agar in
the tube stays at a slant.
Why use a slant?
We can inoculate just the top of a slant to get growth of aerobic bacteria, or we can stab a
needle of bacteria into the tube to see if there are any microbes that can grow without air
(anaerobic). Also, Petri dishes will only keep fresh for 3 weeks and then they dry out.
Tubes will last for a long time in the refrigerator, and are useful for making stock cultures
(pure cultures).
CULTURE MEDIA CLASSIFICATION
1. By Consistency
2. By Contents
a. All-purpose
b. Selective
c. Enriched
Consistency refers to a liquid, solid, or semi-solid. The use of a semi-solid allows us to do two
things: see if the microbe is motile (can move), and to see if the microbe is aerobic or anaerobic.
All Purpose media
Nutrient agar (NA) does not support fastidious organisms (those that are hard to grow because
they need special nutrients), so NA is a good all-purpose media. It will support the growth of a
wide variety of organisms. It is also inexpensive.
Selective media
This type of media selects for a particular type of organism to grow.
An example is Sabouraud’s Dextrose Agar (SDA), which has a high sugar content and an acidic
pH. SDA isolates molds and yeasts, which do well in high sugar content, whereas bacteria are
inhibited. Therefore, sugar acts as a bacterial preservative; that’s why jams, jellies, and preserves
don’t get bacteria growth. However, molds are aerobic and they like sugar. They can get into your
jelly jar when their microscopic air-borne spores drift in whenever the jelly jar is opened. If you
see mold in your jelly, toss out the whole jar because some molds produce cancer-causing toxins!
Molds and yeast also grow well in an acidic environment.
SDA was originally designed to isolate molds of skin, nails, and hair, called dermatophytes,
which are opportunistic pathogens (organisms that only cause disease if the skin in broken, or the
person is diabetic or otherwise ill). Dermatophytes produce ringworm, especially athlete’s foot.
Yeasts are known for causing infections in women, diabetic and cancer patients. Since people
with diabetes have a high sugar content, they are susceptible to such infections. Scrape off a little
skin and place in SDA to isolate the microbe. SDA has 40 grams of glucose and a pH of 5.6.
Enriched Media
This type of media has added nutrients such as vitamins and amino acids. It is used to grow
bacteria which are hard to grow, known as FASTIDIOUS organisms. Many pathogens are
fastidious. An example of enriched media is Blood Agar, which has Trypic Soy Agar (made from
soy) and 5% sheep’s blood (defibrinated to keep it from clotting).
BACTERIAL CULTURES
We may get the bacterial inoculate from a stock culture of a pure organism. We might use an
unknown culture of organisms that you need to grow and run tests on for identification. In either
case, you need to learn to transfer the bacteria from the culture to the sterile medium. The
bacterial inoculate can be transferred using an inoculation loop (a wire with a small loop at one
end and a handle at the other) or an inoculation needle (wire on a handle, but without a loop on
the end). Since these tools are made of metal, they can be used and then re-sterilized in a flame
repeatedly.
CONTAMINANTS
The media on which you want to grow your new culture may also grow undesirable contaminants,
especially molds and other types of fungus, and bacteria from your skin and hair. It is therefore
essential that you protect your cultures from contamination from airborne spores and living
microorganisms, surface contaminants that may be on your instruments, and from skin contact.
Bacteria and other contaminants cannot fly. Nearly all forms of contamination are carried on
microscopic dust particles that make their way onto sterile surfaces when they are carelessly
handled. Therefore, we must maintain the rules of aseptic technique, which decrease the chance of
contamination. Aseptic technique is not as pure as sterile technique. It means that we are using
caution to prevent infection or contamination, but it is not as strict as sterile technique. When a
nurse or doctor cleans an infected wound, the site is considered contaminated already (since it is
infected), so aseptic technique is used. Clinically, using aseptic technique means you don’t have
to scrub the patient’s skin with Betadine (antimicrobial scrub) or use sterile gauze. You also don’t
have to wear a gown or face mask. However, if the patient is having a sterile surgery, sterile
technique is used.
In lab, we are not dealing with wounds, just the organisms that infect them. Using the phrase
“aseptic technique” now refers to being able to safely transfer organisms from one location to
another, without spreading the organism to other locations, and without contaminating a pure
culture that we are trying to grow.
ASEPTIC TECHNIQUE FOR TRANSFERRING BACTERIA
General preparations

Never leave a culture dish open, even for a short time when viewing colonies of
organisms. During your procedure, keep the lid close to the dish, open it only as far and as
long as is necessary to accomplish the procedure, and keep the lid between your face (and
your germs!) and the agar surface.

For most bacterial cultures you will use a sterile loop or needle to inoculate or to obtain an
inoculum. Never reach for a loop or needle to grasp the metal…it might be scalding hot
from a previous student. Always grab the instrument by the handle.

Prepare the equipment: Place the Bunsen burner in front of you and assemble all
necessary equipment with in arms reach. Position everything so that you will not burn
yourself while trying to inoculate your tubes.
Label the tube or plate to be inoculated with the date, the test type, your lab period, and
your name. If it is a tube, place it in a rack in front of you.


Prepare the Bunsen burner by adjusting the flame so the yellow flame disappears and just
the blue flame is visible. Then decrease the blue flame until a blue cone of flame is seen near
the tip of the Bunsen burner. Be careful…this flame is hard to see, so never reach over a
Bunsen burner, since you can never be sure whether it is on or not.

Flame a loop or needle by holding it angled downward into the blue part of the flame.
Start the flame at the loop. When the lower 1/3 of the instrument glows red, push the
instrument further into the flame so the middle 1/3 is in the flame. When that glows red,
push it in further so the upper 1/3 of the instrument can be flamed. It is now sterile. Don’t
let any part of the metal touch any surface until you are ready to obtain your scoop of
bacteria (called the inoculum). Also, don’t flame a loop that is wet, or it will spatter,
scattering bacteria with it!

Let the instrument cool by holding it for 30 seconds. You can make sure it is cool by
touching it to the agar or liquid surface in your culture before you obtain your culture. If
the instrument is too hot, it will kill the culture on contact. While waiting for the loop to
cool, do not wave it around to hasten cooling, and certainly don’t blow on it. Either
action could introduce bacterial contamination.

When the loop or needle is cool (30 seconds), pick up the tube that contains the culture
that you want to transfer, and hold it by the base in your NON-dominant hand. Your
sterile loop is in your dominant hand. Take off the top of the culture tube with your
dominant hand, between your pinky and ring finger….this takes a while to develop the
skill. Never place a lid on another surface. You must hold the lid in your hand during
the transfer. Make sure the open side of the lid is facing downward, so no contaminants
can float down into it. The lids and the sterile loops should be in your dominant hand, and
the culture tube is in your non-dominant hand.

Flame the tube: Pass the neck of the glass culture tube through a flame (two quick, backand-forth strokes), with the container at about a 45° angle toward the flame. Use the blue
part of the flame. This tube should be in your non-dom. hand.

The sterile, cooled loop or needle is in your dominant hand. Stick it into the pure culture
tube, obtain your culture inoculum, then withdraw the loop or needle.
Flame the tube again, then place the lid back on. In the meantime, the pure culture
inoculum is on the loop, being held by your right hand…be aware of it at all times so you
don’t accidentally touch it to your shirt or any other surface, or you will have to flame it
and start over.

Obtaining the inoculum

If you are obtaining the inoculum from a tube, the tube may contain solid agar or
nutrient broth. In either case, you usually use a loop instead of a needle. Hold the
instrument like a pencil in your dominant hand. If it is a broth, shake the tube a little, then
just dip the loop into the broth and the liquid will spread out across the loop. If it is solid
agar, gently scrape the top of the loop across the surface of the culture in the tube. Be
careful not to dig into the agar. If the agar in the tube was solidified in a slanted position,
keep the loop parallel to the agar surface to prevent digging into the medium.
o


To obtain a large inoculum from a broth culture, you need to use a sterile
disposable pipettes, so read your procedure carefully to see when you need to use
this technique.
If you are obtaining inoculum from a Petri dish with a loop, scrape the top of the loop
across the surface of the culture, being careful to keep the loop parallel to the agar surface
to prevent digging into the medium. Remember, don’t take the lid all the way off the Petri
dish, just lift it a little to obtain the sample.
If you are obtaining inoculum from a Petri dish with a needle, just touch the tip of the
needle to a single colony, being careful not to dig into the medium. Don’t pick up more
than one colony. This technique is used when the Petri dish contains multiple types of
bacteria and you want to subculture one pure colony to run tests on it for identification.
Performing the inoculation

If you are inoculating a Petri dish, pick up the lid with your non-dominant hand, and
only raise it a little, keeping the lid face down and directly above the Petri dish. The
inoculum on the loop should still be in your dominant hand. Streak the plate with the loop
(or needle), being careful not to touch the outer surface of the Petri dish. Since both hands
are occupied, the Petri dish base must be left on the table.

Replace the Petri dish lid, then re-flame the loop or needle after performing the procedure,
putting down safely without burning the bench, you, or another student.
o
To inoculate a Petri dish, there are several techniques (streak, streak for isolation,
etc) which will be described later.

If you are inoculating a tube instead of a Petri dish, you must keep the tube in the rack
(since your dominant hand is occupied), take the lid off with the two little fingers of your
non-dominant hand, then pick up the tube also in your non-dominant hand, flame the glass
neck, then inoculate, flame the neck again, set the tube down in the rack, then replace the
lid. Then you can re-flame the loop or needle.

There are so many things to keep track of while doing this! Pay the most attention to the
culture until the sterile Petri dish (or sterile tube) is exposed, then pay the most attention to
keeping the inoculated medium free from contamination until the lid is closed, then pay
attention to sterilizing the loop carefully.
o
To inoculate a broth in a tube, place the loop in the broth, and knock the loop
back and forth across the sides of the tube with the loop is immersed in the broth.
o
To inoculate an agar tube, there are several techniques (streak, stab, etc) which
will be described later.
General considerations

Always be aware of where your hands are, where your face is, and whether or not your
culture is in a position to be contaminated. If you have long hair, make sure it does not
hang into your plate. Hair is full of potential contaminants, and is one of the principle
sources of contaminating microorganisms. The hair or clothes do not have to touch the
sterile surface to contaminate it…bacteria are constantly falling off us, so be sure to keep
the lids face-down, and use the lid to cover the open sterile surface.

If you have an open flame, long hair that is not tied back or loose clothing can be
hazardous to your health.

Keep flammables away from the flames, including alcohol used for sterilizing
instruments; do not place a heated loop or glass rod into an alcohol dish or it will burst
into flames immediately.

One of the most common way to start a fire is to reach over an open flame, and your lab
coat will catch on fire. Take your coat off immediately! While you are taking it off, you
can use the rest of your coat to smother the flames on your arm.

Place test tubes in racks when working at your table: never lay the tubes down--they
leak.
Do not dump ANY microbial suspension down the drain--only in the discard area.
The gas should be turned all of the way on, so that the level is parallel with the rubber
tubing


METHODS OF APPLYING THE INOCULUM
To inoculate the sterile tube or plate, there are different techniques, depending on what tests you
will perform on the subculture. Make sure you use the proper technique.
Streak plate
This is typically used to introduce a pure bacterial sample to a new plate and spread that sample
out sparsely enough to facilitate the growth of distinct bacterial colonies. The inoculum is either
obtained from a tube, or if the sample is from a patient, the inoculum might be on a sterile cotton
swab (throat or wound swab). The inoculated loop or swab is just streaked in a zig-zag across the
plate, from top to bottom.
Streak for isolation
1. Use a Sharpie pen to draw four quadrants on the outside bottom of your Petri dish. Going
clockwise, label each quadrant 1-4.
2. Streak the inoculum in a zig-zag motion over Quadrant #1 from top to bottom. If you used
a patient swab, discard swab in biohazard bag.
3. Sterilize the loop in flame of Bunsen burner.
4. Allow loop to cool without waving it around.
5. Rotate the plate so Quadrant #1 is to the upper left, and Q2 is on the upper right.
6. Place loop on the inside edge of the dish and tap it on the agar a few times to make sure it
is cool. Then touch it into Quadrant #1 and drag one line into Quadrant #2 and streak in a
zig-zag motion throughout Quadrant #2.
7. Again sterilize loop in flame of Bunsen burner, and allow loop to cool without waving it
about.
8. Rotate the plate so Quadrant #2 is to the upper left, and Q3 is on the upper right.
9. Place loop on the inside edge of the dish and tap it a few times to make sure it is cool.
Then touch it into Quadrant #2 and drag one line into Quadrant #3 and streak in a zig-zag
motion throughout Quadrant #3.
10. Again sterilize loop in flame of Bunsen burner and allow loop to cool without waving it
about.
11. Rotate the plate so Quadrant #3 is to the upper left, and Q4 is on the upper right.
12. Place loop on the inside edge of the dish and tap it a few times to make sure it is cool.
Then touch it into Quadrant #3 and drag one line into Quadrant #4 and streak in a zig-zag
motion throughout Quadrant #4.
13. Incubate plate at 37 degrees C for at least 24 hours.
Streak for isolation
INOCULATING ONTO AN AGAR SLANT WITH A LOOP
Place the loop with bacteria into the slant tube, all the way down to the bottom of the slant. You
might be asked to just bring the loop straight up the slant, or to zig-zag the streak as you bring it
up.
INOCULATING ONTO AN AGAR SLANT WITH A NEEDLE
Stab the inoculum down to the bottom of the agar in a clean, straight stroke. Pull the needle out of
the same hole you created when you stabbed it. Sometimes you also streak the top of the surface
with a zig-zag…check your directions for that experiment.
http://www.youtube.com/watch?v=tBmNitxvqyc
INCUBATION
Make sure your plates are labeled on the bottom. Plates are always incubated UPSIDE DOWN
because if condensation forms on the top, we don’t want it to drip onto the culture and ruin it.
DESCRIBING COLONY MORPHOLOGY
Know terms describing types of elevation in colonies, such as erose, umbonate, flat, convex,
filamentous, rhizoid, and lobate. Be able to use these words to describe your colonies.
PIGMENTATION CONFIGURATION
1. orange
round
2. beige
irregular
MARGIN
smooth
erose
ELEVATION
convex
flat
3. white
rhizoid
rhizoid
growth into medium
Negative Stain
NEGATIVE STAIN (Nigrosin)
What makes a stain a negative stain? It is acidic with its negative anion on the chromophore
(color portion of the dye). That means the color portion of the dye has a negative charge. Bacteria
cells also have a negative charge, so the bacteria cells repel the chromophore (dye). Therefore, the
cell will have no color. Just the background is stained.
Negative Stain Prep
With NO gloves on, clean several slides, rinse, then squirt alcohol on the slides and dry with a
paper towel and set them aside on a clean paper towel. Place one SMALL drop of Nigrosin to the
edge of a slide and set aside.
We will be using aseptic technique, so we need the Bunsen burner, gloves, and lab coat.
MOUTH BACTERIA
Clean four slides, rinse, and dry.
Place one drop of Nigosin on the edge of a slide.
Scrape the pointed end of a toothpick between your teeth to pick up some plaque.
Wipe the plaque from the toothpick to the edge of the slide.
Mix the plaque with the stain using the toothpick in a tap-and-mix motion.
Throw the toothpick in the biohazard bag.
Take a clean slide (not a coverslip) and place it at a 45 degree angle facing TOWARD the stain
drop. Slide it into the drop and wait five seconds so the capillary action draws the stain up and
down the edge of the slide.
Maintaining the angle, quickly draw the top slide across the bottom slide to smear the drop. Leave
the slide out to air dry.
The bacteria cells will look clear against a gray background.
You do not need to heat fix this slide, and don’t use a coverslip.
Whenever you look at a slide with oil immersion, you can change the objectives back to one of
the lower two lenses, but do not ever use the hi-dry lens on a slide that already has oil on it, or it
will get oil on the wrong lens.
TAKING PURE COLONY WITH A NEEDLE
Place on drop of Nigrosen in the center of a new slide. Take the sample with an inoculating
needle instead of a loop. A loop picks up too much of the colony and it will be hard to see the
individual bacteria cells. An inoculation needle picks up a much smaller amount of sample, so it
will be easier to see.
Sterilize the needle from the tip to the end of the wire where it attaches to the handle by passing it
through the tip of the inner blue cone of the flame. Cool it by dipping the wire tip into the agar in
a clear area of the plate. What will happen if we touch a hot needle to a colony? It will be
destroyed and change the normal morphology (size, shape, and arrangement of cells). Remember,
when you lift the lid of the plate, hold it above the plate as a shield from air contaminants. Touch
the inoculating needle to one colony and replace the lid.
Touch the inoculating needle tip into the Nigrosin drop and mix well by stirring in circles with the
tip of the needle, but do not enlarge the drop much. Place a spreading slide at 45° to the drop,
contact the drop for 5 seconds and spread the stain across the slide. Sterilize the needle again, and
because it has sticky Nigrosin on it, rinse in water and wipe with a towel before you return it or
use it again. This will remove the Nigrosin that the flame does not remove.
There is no need to heat-fix Nigrosin because the stain makes the bacteria stick to the slide. Just
let it air dry. When your slides are all prepared, you can clean up your area and remove your
gloves. When viewing the Nigrosin slide, observe under 1000x and oil. Observe the colony
morphology (color, margin, elevation) of the pure culture you took a sample from. Also observe
what you see under the microscope, including shape of cells (bacilli, cocci, etc) and arrangement
(staphylo, tetrads, strepto, etc).
Above is a drawing of bacteria with
a negative stain. The most practical
use for a negative stain is to
determine cell size and morphology
(shape) because there is no need to
heat-fix the slide. Heat-fixing
causes the cells to shrink.
This is an actual photo of a negative
stain. See the spirochete in the
center (looks like a wavy noodle)?
This organism is frequently found
between the teeth.
STAIN TECHNIQUES
Making a stained slide of your own cheek cells involves several steps. First, the slide is cleaned
and dried. Then the flat part of a wooden toothpick is used to scrape the inside of your cheek. The
toothpick is then smeared onto the slide and allowed to air dry. If we look at the smear under the
microscope at this point, the cells will be very difficult to see because there is little or no contrast:
they will be almost clear against a bright background. Therefore, stain is applied. Preparing a
smear of bacteria is similar; using a sterile loop, a colony of bacteria is smeared on a clean slide
and allowed to dry before staining.
The first step in preparing a slide for staining is to “fix” the slide. This is done by passing the slide
through a flame a few times. The purpose of this is to attach the cells (or the bacteria) to the slide
and kill the microbes. This procedure shrinks the cells and causes the proteins in the cells to
become like glue. The slide is then stained so they can easily be seen. You must beware of
“artifacts” when you are viewing slides you prepare. They are pieces of dried dye, dust, or other
substances that are not part of the specimen.
The stain is a dye that is made of a salt with a colored ion (called a chromophore). If the ion has a
positive charge it is called a cation; if it has a negative charge it is called an anion. A cation
creates a basic dye (pH higher than 7) and an anion creates an acidic dye (pH lower than 7).
A basic dye is used to stain the cells that you wish to observe. This is the type of stain that we will
mainly be using in lab. An example is the Gram stain. Therefore, Gram stain is a salt with a cation
on the chromophore, and has a basic pH.
An acidic dye, also called a negative stain, is used when you want to stain the background instead
of the cells. An example is Nigrosin, which we used in the last lab. Since there is no heat fixation
with Nigrosin, the cells don’t shrink. Therefore, this is the stain technique to use when you want
to measure the size of cells. We can measure the cells by using a tiny ruler in the eyepiece of the
microscope called an ocular micrometer.
TYPES OF STAINS
There are different types of basic stains. A simple stain uses only one stain; an example is
methylene blue. This is what we will use to stain cheek cells.
A differential stain uses several stains; and example is the Gram stain. This is used to stain
bacteria, to help identify what type of bacteria we are seeing.
There are also a number of special stains for viewing spores, capsules, or flagella.
SIMPLE STAIN (Methylene Blue) Of Cheek Cells
Place one drop of methylene blue on the slide.
Obtain bacteria by scraping the inside of your cheek with the FLAT end of the toothpick.
Place the toothpick with bacteria directly into the stain and spread in circles until the stain is
spread out very thin with no gaps in the circle of stain.
When the slide is completely air dried (if wet, the water will spatter, spreading bacteria), heat fix
the slide by passing it through a flame three times. The purpose of heat fixing is to make the cells
stick to the slide so they don’t rinse off when we apply the stain.
Place the slide on a rack over the sink and cover the smeared area with methylene blue.
Allow to sit for only one minute. Tilt the slide over the sink and use a water bottle to gently rinse
the stain off; make sure the stream of water is on the upper edge of the slide where there is no
bacteria. Otherwise, you might rinse the bacteria right off the slide.
Press the slide in bilbulous paper several times and measure some bacteria.
SIMPLE STAIN (Methylene Blue) OF BACTERIAL COLONY
Take one of your clean slides and mark the underneath surface, in the center of the slide, with a
circle the size of a dime. Sterilize a loop and allow the air to cool it. Place several drops of water
from the water bottle onto the loop until you get a loop full of water. Tap the loop onto the center
of the circle on the slide.
Obtain the colony sample with the inoculating needle and smear the bacteria into the water drop
in progressively wider circles. Air dry completely to avoid heat-fixing a wet slide that will spatter
bacteria (aerosols). The next step is to heat fix the slide. Why do we do this? Two reasons: to kill
the bacteria so there is less danger, and to stick the bacteria onto the slide.
Place one drop of methylene blue onto the slide for 1 minute, rinse gently with water bottle, blot
dry with Bibulous paper, and observe under 1000x.
GRAM STAIN
This is the most common type of stain. It is the first step in identifying an organism. It does not
identify the organism, but it is the first step. It is one of the differential types of stain. The
reaction of stains is different because of differences in the chemistry of the cell wall.
The cell wall of a gram negative organism has many lipids in the cell wall:
LPS – lipopolysaccharide
LP – lipoproteins
PL – phospholipids
GRAM POSITIVE
Purple
Thick peptidoglycan
Very little lipid
GRAM NEGATIVE
pink
thin peptidoglycan
Lots of lipid (in the outer membrane)
The Gram stain is used to distinguish between Gram positive bacteria (will look violet because
they are not decolorized) and Gram negative bacteria (will look pink from the safranin because
they were decolorized). Since all bacteria are either Gram positive or Gram negative, this stain is
the first thing used to determine what type of bacteria is present in the specimen. This helps us
figure out what organism we are dealing with. The results are recorded as Gram positive or Gram
negative.
NOTE: All differential types of stains have the following steps, but with variations in order and
chemicals.
Gram Stain Procedure
First, prepare the sample on a slide, air dry and heat fix.
1. PRIMARY STAIN (the stain used first)
a. Crystal Violet
b. Leave on for 1 minute
c. Rinse gently with distilled water
2. MORDANT (a mordant is something that causes the primary stain to form a complex
with it to chemically react with the cell).
a. Gram’s Iodine (not regular iodine)
b. Leave on for 1 minute
c. Rinse gently with distilled water.
d. The iodine forms a complex with crystal violet which reacts with the
peptidoglycan in the cell wall.
e. Without iodine, all of the stain would wash away.
3. DECOLORIZER
a. 95% Ethyl alcohol (ETOH)
b. As an alternative, you can use ETOH mixed with acetone.
c. This is the most critical step because the alcohol must be left on for just the right
amount of time.
d. Apply the alcohol by dripping it onto the slide, while rocking and rolling the slide
as you hold it.
e. Keep adding the alcohol and WATCH THE WASH for when the purple wash
becomes clear, then STOP.
f. Rinse gently with distilled water.
g. Decolorizing time should be about 5 seconds for a thin smear and 10 seconds for a
thick smear.
4. COUNTERSTAIN
a. Safranin
b. Leave on for 1 minute
c. Rinse gently with distilled water
d. Blot dry.
What are some reasons why you might get a Gram positive culture show up with both purple and
pink cells that are all the same size?
1. The culture is too old (more than 24 hours). Some of the cells have died, and the
peptidoglycan breaks apart, so it appears Gram negative.
2. The decolorizer was left on for too long.
3. The sample smear was too thick and the stain did not get through to all the cells.
Suppose you look at a Gram stain of staphylococcus and you see staphylo, diplo, singles, and
tetrads on your slide. They are all purple and they are all the same size. Is the culture
contaminated? No, it is probably still pure because the cells are all the same size. The clusters just
broke apart during the preparation, or you are seeing a new cell in the process of dividing into a
cluster.
If you see both purple and pink cells of different shapes, that is not a pure culture. If the culture is
supposed to be pure, it became contaminated.
Problems:
When you do a Gram stain:
What would happen if you forget to use Gram’s iodine? (cells would be pink)
Over-decolorize? (cells would be pink)
Use no alcohol? (cells would be purple)
Use an old culture? (Gram + cells would be purple and pink because some PG has broken
down)
GRAM STAIN SUMMARY
1. PRIMARY STAIN: Crystal violet. This is the first stain used.
2. MORDANT: Iodine. The mordant is what allows the primary stain to react chemically
with the cell. It forms a complex with crystal violet and peptidoglycan in the cell wall of
bacteria. It keeps the crystal violet from being washed out by the alcohol.
3. DECOLORIZER: Alcohol or acetone. This removes the primary stain from some of the
cells (decolorizes some of the cells).
4. COUNTERSTAIN: Safranin. This is a red color that stains the cells that became
decolorized.
CAPSULE STAIN: Some bacteria have a capsule which resists phagocytosis (being eaten by our
white blood cells). An example is Streptococcus pneumoniae. This stain colors the background
but the capsule remains clear. This will reveal the presence of a capsule, assisting in the diagnosis.
COMPARISONS OF STAINS
G+ (PURPLE)
PRIMARY STAIN
MORDANT
DECOLORIZER
COUNTERSTAIN
Crystal violet
Iodine
ETOH or
acetone:
Cell is purple
Safranin:
Cell is purple
G – (PINK)
Crystal violet
Iodine
ETOH or
acetone:
Cell is clear
Safranin:
Cell is pink
ACID FAST
CAPSULE
STAIN
Carbol Fuschia Malachite green
Heat
Heat
Acid alcohol
Water
Methylene
Blue
Safranin:
Spores are green
Cell is pink
SPORE OR ENDOSPORE STAIN: When the environment becomes too harsh to survive, some
bacteria have the ability to eliminate all their cytoplasm and condense all their essential DNA and
organelles into a highly resistant structure called a spore, which is metabolically inactive. When
the environment improves, they can re-establish themselves. Only sterilization can kill a spore.
Spores are usually only produced by bacillus bacteria that are found in the soil, such as Bacillus
(non-pathogenic) and Clostridium (tetanus and botulism)
a.
b.
c.
d.
PRIMARY STAIN: Malachite green
MORDANT: Heat (allows dye to penetrate the spore)
DECOLORIZER: Water
COUNTERSTAIN: Safranin
ENDOSPORE STAINS
Endospores are the most resistant cells on the planet. Whatever the conditions are for killing
spores are the conditions necessary for sterilization. The reason we use an autoclave at 121°C for
at least 15 minutes with steam under pressure is because that is what it takes to kill endospores. If
there was no such thing as an endospore, we could just boil everything for ten minutes to get
sterilization.
Don’t confuse endospores with other spores like reproductive spores of fungi. Reproductive
spores are not resistant. Bacterial endospores are not reproductive.
Terminology
Vegetative cell: This is a cell that can make the endospores, but they are not present yet.
Endospores can be inside the cell or free.
Free endospores do not have any cytoplasm left.
Two major genera that produce endospores
1. Bacillus (an obligate aerobe; must have O2
2. Clostridium (obligate anaerobe; must not have any O2)
(There are other bacteria that are aerotolerant aerobes; if they are exposed to air, they won’t die,
but they won’t grow, either.)
Why make endospores? To resist adverse environmental conditions: changes in temperature,
pressure, pH, oxygen, and moisture.
When we inoculate media and place it in the incubator, that’s all the nutrients the bacteria get. No
one comes in and adds more vitamins. When the nutrients are all used up, the bacteria die unless
they can make endospores.
Characteristics of Endospores
1. Highly resistant to adverse environmental conditions
2. No metabolism
3. No water left in the cell
4. They retain their DNA
5. They have a vary thick spore coat for protection
6. Can remain viable (able to return to the vegetative state and reproduce again) for millions
of years
Two Cycles of Endospores
1. Vegetative Cycle
2. Sporulation Cycle
VEGETATIVE CYCLE
Endospore-forming bacteria reproduce by binary fission, just like all other bacteria. Every 15-20
minutes, they split into 2, then 4, then 8, then 16, etc.
Vegetative cells are happy, they have nutrients, and environmental conditions are good.
Therefore, they have no endospores. In a vegetative state, these cells are easily killed by heat or
chemicals.
When the environmental conditions become adverse, the nutrients deplete, the O2 levels either go
up or down (whichever is unsatisfactory for that organism), and the water availability goes down
and becomes dry (dessication). Under such conditions, the vegetative cell will then enter into the
sporulation cycle. We can induce this cycle by taking bacillus and incubating them for 48 hours,
because the nutrients will deplete by then. After 72 hours, there will only be free endospores left.
In 24 hours, we can see vegetative cells with and without endospores, as well as free endospores.
SPORULATION CYCLE
We can see endospores even without stain because they are highly refractive; light from the
microscope bounces off of it.
Spore Locations Within Cell
1. Central
2. Terminal
3. Subterminal
Today, we will use Bacillus spp, which means that we don’t know what species are in there.
These were obtained from your air exposure plates. We know they are not clostridium, because
the incubator allows air to get in.
There are two endospore stain techniques we will use today.
1. SHAEFFER-FULTON SPORE STAIN
Clean two or three slides with Bon ami, rinse, and clean with alcohol. Add one loop of water
to the slide and add a needle sample of Bacillus from the culture tube. Air dry, heat fix, and
prepare the steam heat like we did last lab period. Place a small square of bibulous paper over
the smear, and this time add one drop of malachite green every 30 seconds for 5 minutes.
Endospores and cytoplasm will now be green. You need a spore stain to see free endospores.
Rinse off the stain, and the cytoplasm will be clear. Counterstain with safranin to stain the
cytoplasm.
a. Primary Stain: Malachite green
b. Mordant: Steam heat
c. Decolorizer: distilled water
d. Counterstain: Saffranin
ACID-FAST STAIN
The results of this stain are recorded as acid-fast or non acid-fast. An example is the ZiehlNeelsen stain. Acid-fast bacteria look pink and non acid-fast look blue.
1.
2.
3.
4.
PRIMARY STAIN: Carbol fuchsin (purplish-pink color)
MORDANT: heat
DECOLORIZER: acid alcohol
COUNTERSTAIN: Methylene blue
This is the stain of choice if one suspects an organism with a cell wall made of mycolic acid,
which is a waxy substance that resists Gram stains. The heat in this procedure will melt down the
wax in the cell wall to allow the stain to get in. Two organisms that are acid-fast that are
pathogens (cause disease) are Mycobacterium and Nocardia.
MYCOBACTERIUM
1. Mycobacterium tuberculosis: an air-borne pathogen that causes tuberculosis.
2. Mycobacterium leprae: Causes Hansen’s disease (formerly known as leprosy).
NOCARDIA
1. Nocardia asteroides: lives in the soil. When inhaled, it can cause pneumonia, but usually
only an opportunistic infection in immunocompromised patents.
Opportunistic infections are infections caused by organisms that usually do not cause disease in
a person with a healthy immune system, but can affect people with a poorly functioning or
suppressed immune system. They need an "opportunity" to infect a person. Immunocompromised
patients include elderly people or infants, AIDS or HIV-infection, Immunosuppressing agents for
organ transplant recipients, chemotherapy for cancer patients, malnutrition, medicines (some
antibiotics), medical procedures (surgeries, especially implanted joint replacements or internal
fixation hardware such as screws and plates for broken bones).
ZIEL-NEELSEN ACID-FAST STAIN
The acid fast stain is a differential stain used to identify only two types of bacteria.
The only organisms that are acid fast (the acid-fast genera) are:
1. Mycobacterium
a. Causes Hansen’s Disease (leprosy)
b. Causes Tuberculosis (TB)
c. There are also non-pathogenic species
2. Norcardia (opportunistic pathogens; only cause disease in those with poor immune
systems, etc).
The thing that makes organisms acid fast is the wax (mycolic acid) in their cell walls.
Their endospores resist stain because of mycolic acid.
Clinically, it is important to be able to identify these organisms quickly. This test is only used
when a patient is suspected of having TB or leprosy. It is especially useful when someone is
suspected of having TB; the sample is obtained from the sputum, and an acid-fast stain is
performed to give a preliminary diagnosis right away. It can also be performed on patient samples
to track the progress of antibiotic therapy and determine the degree of contagiousness. There are
10 million new cases of TB per year, 3 million deaths, and it affects 1/3 of the world’s population.
The results are recorded as AF (acid fast) or NAF (non-acid fast). Don’t record them as positive or
negative (like the lab manual says) or you may get them confused with Gram stain results.
In the SF stain technique, the primary stain is applied with heat (steam). The reason for this is that
the heat increases solubility of the mycolic acid so it can react with the primary stain. Therefore,
heat allows stain to penetrate resistant cells because mycolic acid is waxy. Blotting paper must be
used on top of the stain when heat steam is used; it keeps the stain from drying out.
We will be preparing a slide that is a mixture (emulsion) of two organisms:
1. Mycobacterium smegmatis (AF)
2. Staphylococcus aureus (NAF)
Mycobacterium smegmatis is not pathogenic (we are only SL-2 here). It is saprobic. That means it
lives off dead organic matter. It is part of the normal microbiota of our skin and the oils in our skin. It
also likes dirt. So if you don’t wash regularly, this organism will thrive. It especially lives on the
external genitalia: under the foreskin of uncircumcised males and the labia majora of females. It
produces a cheesy substance called smegma, which has a foul odor. It only takes one day without
washing for it to grow. Hospitalized patients nowadays are in such bad shape they can’t take care of
themselves very well, so they frequently get smegma. You’ll learn to recognize the smell!
With the ZN technique:
1. Primary stain is carbolfuscian (lipid soluable; can penetrate waxy cell wall). It will stain
both AF and NAF.
2. Mordant is the steam heat
3. Decolorizer is ACID alcohol. This will rinse the color out of the NAF only.
4. Counterstain is methylene blue. This will be taken up by the NAF cells.
Ziel-Neelsen Acid Fast Stain Technique
1. Get out a hot plate (the kind that has a coil) and set it on high.
2. Get a metal beaker from your tote box and fill it half way with tap water; place on the hot
plate.
3. Place a wire stain rack on top of the beaker.
4. Place a wire mesh square on top of the stain rack.
5. Clean one slide.
6. Prepare an emulsion:
a. Place one loopfull of water on the slide.
b. Remember to grasp the culture tubes by the glass, not the cap!
c. Add one needle sample of Mycobacterium smegmatus. This organism is very
waxy, so you have to tap and mix VERY WELL to break it up completely.
Otherwise, it will clump on your slide. Page 92 in your manual shows a slide that
is clumpy.
d. Add one needle sample of Staphylococcus aureus and tap and mix.
7. Air dry completely to avoid aerosols.
8. Heat fix.
9. Cut a SMALL square of bibulous paper that is the size of your smear. Place this square
directly on your slide. This keeps the smear from drying out.
10. When the steam starts showing from the beaker, place the slide on the wire mesh and add
ONE DROP of carbolfuscian (the primary stain) to the paper every 30 seconds for five
minutes. Too many drops will cause the carbolfuscian to drip into the water beaker and
boil. This will release phenol from the stain, which is a dangerous aerosol. After five
minutes, turn off hot plate.
11. When the slide is cool, pick up slide with a clothespin.
12. Throw out bibulous paper into the regular trash container.
13. Rinse gently with distilled water.
14. Decolorize with acid alcohol (in the stain kit). Do NOT use regular alcohol! Apply the
acid alcohol with a rock-and-roll agitation for a few seconds until the color rinses clear.
15. Rinse gently with distilled water to stop the decolorization process.
16. Take the slide to the sink and counterstain with methylene blue for one minute.
17. Rinse gently with distilled water.
18. Blot dry.
19. Observe under 1000x and oil.
20. Look for individual cells. You will see AF rods that are purple-pink, and little blue cocci.
This mixture is what you would actually see clinically, because a patient will not have a
pure culture.
What does a slide of ZN look like?
What does a slide of SF look like?
Motility
HANGING DROP PREPARATION
Supplies: Toothpick, petroleum jelly (Vaseline), cover slip, depression slide, gloves
1. Spank the bottom of the nutrient broth to mix it.
2. Remove one loop of broth and touch it lightly to the center of the coverslip, which is
sitting on a paper towel. Try to get the smallest drop possible.
3. Use the toothpick to apply one small dab of Vaseline to each corner of the coverslip. This
keeps the coverslip secure when it is upside down.
4. Clean the depression slide and press the open well down gently onto the coverslip.
5. Gently flip the slide over so the drop hangs in the depression well.
6. Since we will look under the microscope at live cells, there will be no contrast, so turn the
iris down to increase the contrast.
7. Observe under scanning (40x); position the slide so that the edge of the drop is in the
center of the field. Focus on the edge of the drop.
8. Increase to LP then HP, but do NOT go to 1000x because the depth of field at 1000x is too
small to observe the depth of the droplet.
9. Look for tiny specks that are moving and record what type of motility is present.
Problems with hanging drop slides:
False positive results: the organism appears to move, but it cannot. This can happen if you are
observing it too long under the hot light, and the liquid starts to evaporate and recede.
False negative results: the organism is capable of motility, but it does not appear to be moving.
No motility could be observed if the drop falls into the well and you don’t realize it, or if the iris
diaphragm is opened too wide. Motility could be observed without false results if the culture is
too concentrated.
MOTILITY TYPES
1. TUMBLES: the bacterium seems to be rolling over itself like a rolling stone.
2. RUNS: the bacterium moves from point A to point B
3. JIGGLES: the bacterium jiggles like it is in an earthquake, but it does not move from one
part of the slide to another. THIS IS BROWNIAN MOTION, WHICH IS NOT TRUE
MOTILITY.
BROWNIAN MOTION is caused by water molecules hitting a cell with low mass. It causes
movement of low mass cells by the inertia created by molecular bombardment of water
molecules.
C. MOTILITY DEEP
This is a semi-solid agar. Instead of the usual 1.5% agar, motility deeps are 0.5% agar, which is a
liquid gel. It allows the organism to move around, but only along the line of inoculation. This
method of evaluating motility is not as accurate as observing a hanging drop. Evaluate your
motility deep and record the growth pattern.
1. SPREADING GROWTH PATTERN indicates motility.
2. NON-SPREADING GROWTH PATTERN indicates non-motility.
STANDARD PLATE COUNT
How do we count bacteria? There are many methods. Machines like a spectrophotometer can
measure turbidity and thereby estimate the amount of bacteria in a sample. Coulter Cell Counters
can count individual cells as they pass by a light beam. Standard Plate Count is a manual method,
also known as a population count.
Practical Applications
1. Medicine
a. Diagnosing infections: a normal urine sample is expected to have 0-300 bacterial cells
per ml. If the patient has more than 1000 cells it indicated infection. Bacteremia is the term for
bacteria in the blood that are not multiplying. Septicemia is the term for bacteria in the blood that
are multiplying. To determine the difference, we would need to do plate counts from a specimen
drawn on a later date.
2. Food Industry
a. Quality Control: All foods have some bacteria. The County Health Department has
decided what is an acceptable amount of bacterial cells per sample. A food (milk, hamburger, etc)
is labeled “Grade A” when it has less fewer than the allowed amount of bacteria. The County
does random, unannounced testing on samples.
Plate Counts are not qualitative because they do not tell you what organism is present. However,
it is quantitative because it gives you the number of organisms present.
Counting always involves math.
Try to guess how many bacterial colonies are present in a flask that is turbid from E. coli.
Write your guess down in Standard Scientific Notation.
EXPRESSING NUMBERS IN STANDARD SCIENTIFIC NOTATION
For practice: How many zero’s are there? That is the number for your exponent.
1 = 100 = 1.0 x 100
10 = 101 = 1.0 x 101
100 = 102 = 1.0 x 102
1000 = 103 = 1.0 x 103
10,000 = 104 = 1.0 x 104
Now that you’ve had practice, let’s use a random number.
Just count how many digits there are after the first one to get your exponent.
Then round off the given number to the nearest 10th (2 digits):
10634 = 1.1 x 104
Negative exponents (10-1) are fractions less than one.
Positive exponents (101) are whole numbers greater than one.
Now let’s practice using negative exponents:
Round off the third number you come to (other than zero).
For instance, if the number is 0.00342, round down the number to 0.0034
If the number is 0.00345, round up the number to 0.0035
How many digits are there to the right of the decimal point before you get to a number greater
than zero? That is your exponent number.
10-2 = 1.0 x 10-2
0.00126 = 1.3 x 10-3
0.000542 = 5.4 x 10-4
PLATE COUNT PROCEDURE
Remove a set volume of the sample with a pipette. A pipette is an instrument that measures and
delivers an accurate volume. Place the set volume sample in an empty Petri dish, add molten
nutrient agar, and mix. The E. coli have flagella and are motile, so they will spread out a bit;
however, they still tend to aggregate in clusters a bit.
Each group of cells (aggregate) forms a colony. These colonies are called Colony Forming Units
(CFU). A CFU is a cluster (aggregate) of cells which eventually leads to the formation of a single
colony. However, one single cell could also form a colony by itself. Therefore, by doing a Plate
Count, we are unable to determine how many cells are present; we only count the colonies.
Suppose we transfer 1 ml of the E. coli sample to the Petri dish, and suppose that the correct
amount of bacteria present in the solution is 2.5 x 109. That would mean there would be 2.5
billion colonies on that plate. That would be impossible to count!
COUNTABLE PLATE: 30-300 colonies
TMTC (Too many to count): 300+ colonies
TFTC (Too few to count): less than 30 colonies
To get our sample down to the number of colonies that we can count, we have to take less than 1
ml of sample. The pipettes we have can deliver 1 μl of sample, which is 1/1000 ml.
That would give us 2.5 million CFUs on the plate; still too many. Therefore, when the sample is
too concentrated, we need to do a dilution series. But how much to we dilute? 250 CFU is a
countable number.
Micro Lab Unit 2 Notes
PURE CULTURE EXPERIMENT
When a doctor takes a swab from an infected wound and sends it to the lab, he needs to know
what organisms are in the wound so he can select the proper antibiotic. There usually will be
more than one organism present, so the lab will need to take the mixture of bacteria and separate
them out into pure cultures before they can identify each organism. In this experiment, we will
take a mixture of 3 organisms and separate them out into three pure cultures, one of each
organism. Here are the three organisms we will use:
E. coli (forms white colonies)
Serratia marcescens (forms red colonies, but only at room temperature, 25°C)
Chromobacterium violaceum (forms purple-blue colonies, but only at room temperature,
25°C).
All of these organisms are Gram negative, so if you only did a Gram stain on the tissue swab, it
would look like there is only one organism present. Therefore, you have to grow them on a plate
so you can examine the colony morphology (color and shape) of the colonies, and then in real life,
the lab would also run some tests on each pure culture to identify each organism. We already
know the organisms in the mixture, so we will just use this experiment as practice for separating a
mixed culture into pure cultures. You will need this skill when you get your unknown organisms
in the next lab unit.
Streak for isolation
First, draw four quadrants on the bottom of a Petri dish. Sterilize a loop and take one loopful of
the mixture from the flask. Zig-zag the loop over the upper left quadrant of the plate. Sterilize the
loop again, and drag the loop once across the upper left quadrant and into the upper right
quadrant, then (without lifting the loop) zig-zag throughout the upper right quadrant. Sterilize the
loop again, drag one the loop in one line from the previous quadrant and drag it into the lower
right quadrant, and zig-zag that area. Sterilize the loop and drag the last line into the last quadrant
and zig-zag. Then let your plates grow until the next lab period.
When you use this technique, you are dragging fewer and fewer organisms into each quadrant, so
that the last quadrant will have individual colonies that do not overlap each other. If you were
successful, in the next lab period you will see your red, white, and blue colonies as separate
colonies in the last quadrant. It will be easy to tell them apart because we chose organisms that
form different color colonies. If you were not successful, your red, white, and blue colonies will
still be overlapped in the last quadrant. What might have gone wrong? Perhaps you had too
many organisms on the first quadrant, or maybe you dragged too long of a line from one quadrant
to the next, or perhaps your zig-zags were too narrow and did not cover enough surface area.
Incubate your plates upside down, so the agar is on the top. Why do we do this? It prevents
condensation from developing on the inside lid, and then dropping onto your agar, mixing the
organisms. It also makes it easier to retrieve the plate from the sleeve container. If you do not put
the plate in upside down, and you try to lift it from the sleeve container, you might accidentally
lift the lid off your dish, exposing the agar to the air and contaminating it. Keeping the plates
stored upside down also enables you to read the important writing on the bottom of the plate.
After you examine your plate next week, select the very best looking white colony which is
separated from the other colonies, touch it with a sterile needle, and zig-zag it across the surface
of a slant. Do the same for each of the other two color colonies, so you end up with three slants,
one of each organism. You will then let those grow until the next lab period. You will know if
your isolation technique was successful if there is only one color in each tube.
ENUMERATION OF BACTERIA
Sometimes, an emergency room contacts the Center for Disease Control (CDC) to let them know
that they had an unusual amount of people come in with the same illness. Let’s say there is a
sudden outbreak of the stomach flu. The CDC interviews each patient to find if they all went to
the same location right before they got sick. Let’s say that these patients all went to the same
public park. The CDC then examines the park and makes a list of all the possible places that
organisms might be, such as a food vendor, a lake, picnic tables, water fountains, and bathrooms.
They then ask the patients to tell them everything they touched while at the park. Let’s say the
CDC found that everyone who got sick drank out of the same water fountain. They think the
fountain might be contaminated by something, perhaps a broken sewer line underground. They
send a sample of the water to the lab, and they want to know how many organisms per ml there
are. Since drinking water is not sterile, there are guidelines as to how many bacteria are within
acceptable limits. We need to determine how many organisms are present in the sample. We need
to tell the CDC how many living organisms are present in the water, and how many living plus
dead organisms are in the sample.
1. To measure how many living and dead organisms per ml there are in the sample, we
measure the turbidity (cloudiness) by placing it in a machine called a spectrophotometer.
This is considered to be an indirect method of enumerating bacteria, and relies on the
cloudiness of the sample.
2. To measure how many living organisms per ml there are in a sample, we plate them and
count the colonies that grow. This is called a Standard Plate Count (SPC). This is
considered to be a direct method of enumeration of bacteria. Before we can perform a
SPC, we need to perform a serial dilution of the original broth culture.
1. TURBIDITY MEASUREMENT
To measure turbidity, we use a spectrophotometer. You need to understand how this
machine works. This machine sends a beam of light through a tube of liquid sample, and
reads how much light comes out. If we send 100% of light through a tube of clear water, we
will get 100% of light out, and the machine will read 100% transmission. If our sample is not
clear because it contains organisms, we will get something less than 100% transmission. The
light that is not transmitted is absorbed. If 80% of light is transmitted, 20% is absorbed.
Transmission + Absorption = 100%. Therefore, transmission and absorbance are inversely
related. The spectrophotometer has a button that you can set for either transmission or
absorption. Make sure it is set for transmission.
You will place your samples into special tubes called cuvettes, which are test tubes of
optically pure glass that will not absorb light like regular glass test tubes. Place the cuvettes
into the spectrophotometer, and measure the transmission in each sample.
Since our original sample is in nutrient broth (a brown color), the color particles in the broth
will deflect some light, lowering the light transmission. We need to remove that factor so we
can determine only how much light is being deflected by the organisms in the broth. To do
this, we prepare a “blank”, which is a cuvette of sterile broth (the cuvette with a lid on it),
place it in the spectrophotometer, and then set the machine to zero. That will tell the machine
to ignore the color of the broth. How does putting the blank in and zeroing the machine
work? If we put the blank in but do not set the machine to zero, the machine will not know
that this is our “blank”, and it would give us a transmission reading, let’s say 85%
Transmission. That means that the color of the broth is reflecting 15% of the light. If we then
put our sample in, the transmission of our first sample might be 60% Transmission. However,
the correct transmission of our sample should have been 60 + 15 = 75% transmission.
Without using the blank and setting the machine to zero, our readings will be lower than
they should be. When we put the blank in and set the machine to zero, instead of reading
85% transmission, it will now read 100%. Now you are ready to put your first sample in the
machine, obtain a transmission reading (it should be between 1-99%), place your next sample
in the machine (don’t need to blank and zero it anymore), obtain the reading, and continue
until you are done.
Once you have your transmission readings, turbidity is recorded as a sample’s optical
density. To calculate optical density, we use a formula that uses the log of the transmission
reading. Therefore, the next step is to find the log of our transmission reading.
HOW TO FIND THE LOG OF A NUMBER
Download and print the log table from BlackBoard. The log of a number consists of one digit
to the left of a decimal point, and 4 numbers to the right of the decimal point. The digit to the
left is called the character. The numbers to the right are called the mantissa.
If your transmission reading was 60.3, look for the number 60 on your log table, on the left
side of the page (one side has numbers 10-54, the flip side has numbers 55-100). Once you
find the number 60 on the left, slide over to column 3 (because that is the number to the right
of the decimal point of your transmission reading) and find the four-digit number listed there.
That is your mantissa number (7803)
The character is the number of places to the left of the decimal point, minus one. So, to find
the character, look at the number of digits to the left of the decimal point in your transmission
reading. For a transmission of 60.3, there are 2 digits to the left of the decimal. Take that
number of digits (2) and subtract one from it (2 -1 = 1). Therefore, “1” is the character.
That means that the log of 60.3 is 1.7803
Let’s practice:
Problem: Find the log of a transmission reading of 75.8%T
Solution
Mantissa = 8797
Character = 1
Log of 75.8 = 1.8797
Notice that your log table starts at the number 10. What if your transmission reading is less
than 10? The log of 9 is the same number as the log of 90, except the character is 0 instead of
1. The log of 8 is the same number as the log of 80, except the character is 0 instead of 1. So,
if your transmission reading is less than 10, move the decimal point over one place to the right
and find the mantissa for that number instead, and make your character a 0.
Let’s practice”
Problem: Find the log of a transmission reading of 8.9%T
Solution
Mantissa of 89 = 9494
Character = 0
Log of 8.9 = 0.9494 (because the log of 89 is 1.9494)
Once you have the log of your transmission number, we have to calculate the optical density
(OD) for our sample, which will be our turbidiy measurement.
FORMULA FOR OD
2 – log %T
If our transmission reading was 75.8%, the log was 1.8797
OD = 2 – 1.8797
OD = 0.1203
If our transmission reading was 8.9%, the log was 0.9494
OD = 2 – 0.9494
OD = 1.0506
Problem:
What is the OD of 6%T?
Solution
The log of 6 = 0.7782
OD = 2 – 0.7782
OD = 1.2218
Optical Density (OD) is a measurement of the transmission of light passing through a
liquid sample. It is the total amount of light transmitted in a sample that contains both
living and dead bacteria. Notice that the cloudier (more turbid) our sample is, the lower
the transmission (inversely related). Low transmission (and high OD) means there are a
lot of bacteria in the sample. High transmission (and low OD) means there are few
bacteria in the sample. Therefore, OD and transmission are inversely related. We don’t
report the transmission, just the optical density.
When the CDC sees our preliminary report that the water sample from the drinking fountain
has a high optical density, they will be suspicious that this is the source of the outbreak of
stomach flu. A high optical density indicates that there are many particles in the water, but it
might not be a dangerous situation, because the particles might be harmless mineral deposits.
To find out if the cloudy water is from living organisms, we will have to grow them on a plate
and count the number of colonies. Why didn’t we just do this in the first place? We can do the
turbidity test in a few minutes to give the CDC a quick screen test. The plate count takes a few
days to grow.
2. STANDARD PLATE COUNT (SPC)
We will take a loopful of the water sample, put it on a plate, let the bacteria grow for a few days,
and count the number of colonies. To count the number of colonies on a plate, we need to have a
plate that does not have too many colonies to count. To make such a plate, we need to dilute the
original first. Since we don’t know how much to dilute it, we do a series of dilutions (a dilution
series), then plate each dilution we make, and see which plate grows the number of colonies that
are within our ability to count. The number of colonies that are within our ability to count are
30-300. Therefore, we will plate a dilution series, let the bacteria grow for a few days, and then
see which plate has 30-300 colonies. That will be the only plate we will count.
We will take 1ml of our original water sample and add it to a bottle that contains 99 ml of sterile
nutrient solution, and label that Bottle A. Therefore, Bottle A will contain a 1:100 dilution of the
original. If we plate from this bottle, it will grow too many colonies to count. Therefore, we will
take 1 ml from Bottle A and place it in Bottle B, which also contains 99 ml of sterile nutrient
solution. Therefore, Bottle B will contain a 1:10,000 dilution of the original. DO NOT PLATE
BOTTLE B UNTIL AFTER YOU HAVE USED IT TO INOCULATE BOTTLE C. We are
not sure if Bottle B will grow too many organisms to count, so we’d better make another dilution.
Add 1 ml of Bottle B to Bottle C, to make a 1:1,000,000 dilution.
After all three bottles are prepared, you can discard Bottle A.
Label 2 Petri dishes for Bottle B and two dishes for Bottle C.
PREPARING TWO PLATES FROM BOTTLE B
Use a pipette to remove 1.0 ml from Bottle B and put it in one Petri dish. Then pour a tube of
melted agar on top of it, swirl gently to mix.
Use a different pipette to remove 0.1 ml from Bottle B and put it into the second Petri dish. Then
pour a tube of melted agar on top of it, swirl gently to mix.
PREPARING TWO PLATES FROM BOTTLE C
Use a pipette to remove 1.0 ml from Bottle C and put it in one Petri dish. Then pour a tube of
melted agar on top of it, swirl gently to mix.
Use a different pipette to remove 0.1 ml from Bottle C and put it into the second Petri dish. Then
pour a tube of melted agar on top of it, swirl gently to mix. We will then let the plates grow for a
few days to see which plate has 30-300 colonies, and we will count them and calculate the
number of organisms per ml in the original solution.
Each dilution in our series is a one hundred fold (1:100) dilution, but compared to the original, the
last dilution will be one to a million (1:1,000,000). We already know Bottle A will not be dilute
enough, so there will be too many colonies to count, so we don’t plate from the first dilution.
Let’s say the below list is how many colonies we count on what was plated from the dilution
series. Which of the below four plates will we use to count colonies? Discard the other three
plates.
Bottle B, first plate = 1:10,000  800 colonies
Bottle B, second plate = 1:100,000  200 colonies
Bottle C, first plate = 1:1,000,000  20 colonies
Bottle C, second plate = 1:10,000,000  10 colonies
Counting is only significant if the colony count is 30-300 colonies per plate. If there are more
than 300, some colonies may overgrow on other colonies, and the count is not accurate. If there
are less than 30, there are not enough to count. Which of your plates is in that range? Use ONLY
that plate to calculate organisms per ml.
CALCULATE THE NUMBER OF LIVING ORGANISMS IN A SAMPLE
After you count the number of colonies in your appropriate plate, we need to multiply that
number by the dilution factor to see how many organisms were in the original.
FORMULA:
Concentration = Colony numbers x Dilution Factor
Which plate do you use to apply this formula?
We have to use second plate (bold above) because that is the only plate in the 30-300 range.
Solution:
200 x 100,000 = 20,000,000
Covert to scientific notation and write units (org/ml)
2.0 x 107 organisms/ml
That is the number of living organisms were present in the original sample.
Problem:
We counted 50 colonies on a plate that has 1:1000 dilution. What is the total number of organisms
in the original solution?
Solution:
50 x 1000 = 50,000 org/ml
5.0 x 104 org/ml
In your lab manual, there is a graph for you to plot your OD against the dilution factor.
Suppose you know the original concentration of a sample and you want to know the concentration
of the dilutions:
Problem:
The original concentration of a sample is 4.85 x 104 org/mL. This sample was diluted to 1:2.
What is the concentration in the dilution? Answer: 4.85 x 104 ÷ 2 = 2.43 x 104 org/mL
What is the concentration of a 1:4 dilution of the original? 4.85 x 104 ÷ 4 = 1.21 x 104 org/mL
What is the concentration of a 1:8 dilution of the original? 4.85 x 104 ÷ 8 = 6.06 x 105 org/mL
What is the concentration of a 1:16 dilution of the original? 4.85 x 104 ÷ 16 = = 3.03 x 105
org/mL
Remember to download the homework problems, study guide, and the PPT on how to solve
these homework problems. Those types of questions will be 25% of the next lab exam.
EFFECT OF pH ON GROWTH OF BACTERIA
pH measures the concentration of hydrogen ions (H+). The more H+ ions, the more acidic
the substance is, which means it has a low pH (below 7). The less H+ ions, the more basic
(or alkaline) the substance is, which means it has a high pH (above 7). Substances with
neutral pH (like water and much of our body fluids) are pH 7.
The pH scale runs from pH 2 (acid) to pH 14 (base). Each unit change in pH represents a
10 fold difference in H+ ions. That means that pH 3 has 10x more or less H+ ions than pH 2
or pH 4. Since acids have more H+ ions, a change from pH 3 to pH 4 represents a 10x
decrease in H+ ions. A change from pH 3 to pH 2 represents a 10x increase in H+ ions.
A change from pH 3 to pH 5 represents a 100x decrease in H+ ions. A change from pH 3 to
pH 6 represents a 1000x decrease in H+ ions. What is the difference in going from pH 10 to
pH 2? Answer: Subtract 2 from 10 and get 8. That is the number of zeros to place after the
one: 100,000,000. Then say whether it is an increase or decrease. If you go from a high pH to
a low pH, it is an increase in H+ ions. Therefore, going from pH 10 to pH 2 represents a
100,000,000 increase in H+ ions. Likewise, going from pH 2to pH 10 represents a
100,000,000 decrease in H+ ions.
Some organisms prefer a particular pH. If they are placed in a substance that is suboptimal
(meaning less than the best for them), the proteins in their cell walls of the vegetative cells
can become denatured (damaged).
Organisms that grow best at pH 2 and 4 are acidophiles
Organisms that grow best at pH 7 are neutrilophiles
Organisms that grow best at pH 10-12 are alkaliniphiles
We will use the below organisms to inoculate a series of pH tubes, let them grow for a few
days, and use the spectrophotometer to measure growth, and then calculate their optical
density. The tubes with the highest OD (and the lowest transmission readings) contain the
most bacteria. If you sit in the center section of the room, you are Deck 2. To the left is Deck
1, and to the right is Deck 3. Each deck will use one organism, but work in pairs to inoculate
the pH tubes.
Deck 1 has Alcaligenes faecalis
Deck 2 has Saccharomyces cerevisiae
Deck 3 has Staph aureus
Alcaligenes faecalis is an opportunistic pathogen, meaning that it only causes infection if it
has an opportunity to invade. This organism causes urinary bladder infections, so a patient
with a catheter might be at risk; the catheter provides the opportunity for the organism to get
in. This bacterium degrades urea to make ammonia, which increases the pH. Therefore, this
organism is an alkaliniphile.
Saccharomyces cerevisiae is a yeast which is used to make beer. Many yeasts and fungi use
fermentation as a metabolic pathway, and acids are produced in the process. This organism is
an acidophile.
Staph aureus is a resident organism (lives on our skin without causing disease) which is also
an opportunistic pathogen. If we get a cut, it can take the opportunity to invade and cause the
wound to become infected. It is a neutrophile.
At the next lab time, get your pH tubes back, and notice that the colors in each tube are
different. Some are dark yellow, light yellow, dark brown, light brown. Select the first of
your samples to place into the spectrophotometer. Examine the color of your sample, then
examine the colors of the series of blanks in the rack by each spectrophotometer. Select the
blank that most closely matches the color of your sample. Use that blank to zero the machine,
then get the transmission reading of your first sample. To get your sample into the cuvette,
just pour it in carefully. Remember to make sure the reading says %T instead of Abs. Then
select the blank with the color that matches your next sample, zero the machine again, and
get your next transmission reading. Make sure all of your transmission readings are less than
100%. If they are not, first make sure that the machine is set on transmission (%T) instead of
absorbance (Abs). If the machine is set correctly but you still have a reading greater than
100%, just record it as 100%. After you have all of your transmission readings, write them in
black on the board, in the box for each pH listed. Once the entire class has written all their
transmission readings on the board, we will calculate the class average for each pH tube and
write the average in red. Use that red average number to calculate OD for each organism at
each pH. Which organism had the highest OD for pH 2? That organism grew best in
that pH, so it was an acidophile. Which had the highest OD for pH 12? That organism
grew the best in that pH, so it was an alkalinophile. Continue this pattern for all the pH
tubes for all three organisms. Be ready to recall which organism was an acidophile, which
was a neutrophile, and which was an alkainophile.
EFFECT OF UV LIGHT ON GROWTH OF BACTERIA
What is effect of ultraviolet (UV) light on different types of bacteria? The DNA of bacteria
contains two adjacent thiamine’s. UV light causes a bond to form between the two
thiamine’s, and this stops DNA replication. This is the cause of death in the organism. If
an organism can produce endospores, it could survive UV light exposure for a longer
time. In this experiment, we will expose plates of two organisms to UV light for various
periods to see how long it takes to kill bacteria with UV light.
Staph aureus is exposed to a various number of seconds of UV light
Bacillis is exposed to a various number of minutes of light. Why are we exposing this
organism to UV light for a longer period? Bacillus endospores, so it survives longer.
Work in pairs, each pair signs into one of these boxes on the board to choose how many
seconds or minutes your plate will be exposed to the UV light under the hood.
Use a swab instead of loop to swab the plate. Swab the entire plate from top to bottom,
making sure there are no spaces between your zig-zags…you want the entire agar surface to
be swabbed with organisms. Then turn the plate ¼ turn, swab again from top to bottom, turn
¼ turn again, swab from top to bottom, turn ¼ turn again, swab from top to bottom. This
technique is called “Streaking for confluence”. That will give a good spread of organisms
throughout the plate. Use a sharpie pen to draw a line down the center on the bottom of the
plate. Label one half of the plate with “covered” and the other half with “exposed”.
When it is your turn, you will take your plate to the hood. Take an index card and write your
initials on it. After your plate is under the hood, the lid will be removed and placed under the
plate. Then your index card is placed over the half of your plate labeled “covered”. Your
initials on the card should be face up so you can see them. If you have the control plate, do
NOT remove the lid, and do NOT put the index card on. Once you are done with your
exposure time, take the card off, put the lids back on under the hood, and put them in the
sleeve container on the back deck until the next lab period.
The first people to use the hood will be the 60 minute and 30 minute crews. You can all
inoculate the plates now, but wait your turn to use the UV light. Who signed up for the 60
minutes? You go first. Then the 30 minute people go next. That way it is fair; no one gets to
go home early!
Next lab period we will collect the data on the UV light plates. Count the colonies on the
exposed side so you can determine which organism was more resistant to the light. Bacillus
should be more resistant because it makes endospores. Everyone in the class should write
their data on the board. Note the time at which no colonies were found for each organism. If
there are too many colonies to count on your plate, write TNTC (too numerous to count).
Don’t throw out your UV plates. There might be a problem when we look at the data, and I
want to see if I can figure out what the problem is by looking at your plates. Your lab manual
asks you to enter a number that measures bacterial resistance to UV light.
FORMULA TO MEASURE RESISTANCE TO UV LIGHT
Time to kill Bacillus ÷ time needed to kill Staph.
Note: to apply this formula to another two organisms, Divide the longest time by the shortest
time. Suppose the more resistant organism took 30 minutes to kill, and the other organism
took 10 minutes to kill. 30 ÷ 10 = 3. That means organism 1 is 3x more resistant to UV light
than organism 2. It also means that organism 2 is 1/3 as resistant to UV light than organism
1.
EVALUATION OF ANTISEPTICS AND DISINFECTANTS
We need to grow some colonies for the antiseptic experiment, in which we will test the
effectiveness of several different antiseptics. An antiseptic is used on tissue, not inanimate
objects. There are two kinds of antiseptics: topical (like Bactine) and oral (like Scope
and Listerine). We will use colonies grown from your own oral cavity to conduct these
experiments. In the next lab period, you will each test the antiseptic products on your own
bacteria, so you can see the effectiveness of these products on your particular microbes.
For today, just collect your own oral organisms. Take a sterile swab, and swab the inside of
your cheek. Place the entire swab in the TSB tube. Break off the excess wood handle and let
the swab drop in. Work individually, so each person has one tube.
At the next lab period, we you will use your own mouth organisms to swab a plate for
confluence and then apply the antiseptic discs to the plate. Each of you get your own plate
and use your own mouth organisms.
Then work in pairs to swab another plate for confluence, using the one organism assigned to
your deck. This plate will receive the disinfectant discs. Disinfectants are chemicals that
are used on inanimate objects only. This blank paper discs are sterile, and contained in a
sterile Petri dish. When obtaining these discs, use aseptic technique (flame the forceps in
your drawer, just lift the lid of the disc containing the sterile blank discs). You need to apply
the disinfectant or antiseptic to the disc and place the disc on your inoculated plate. When
you prepare your antiseptic and disinfectant discs, make sure the disc is not too saturated
with the product, or it will diffuse into the media, and nothing will grow. To prevent this, just
dip the paper edge into the agar and let capillary action draw it up. Next lab time, measure (in
mm) the clear zone around each filter paper, from the edge of the disc to the end of the clear
zone. For antiseptics and disinfectants, the products with the largest zones of inhibition
are the ones that are the most effective. Note that this is NOT the case with antimicrobials,
where the largest zone is not necessarily the best.
EVALUATION OF ANTIMICROBIALS
Antimicrobials are either narrow spectrum or broad spectrum. Narrow spectrum antimicrobials are
effective against Gram positive organisms because their mechanism of action is as a cell wall
inhibitor, which does not cause a problem for Gram negative organisms. Broad spectrum
antimicrobials kill both Gram positive and Gram negative organisms because the part of the
cell that they damage (e.g. ribosomes) is present in both types of organisms. However, you need
to be careful with broad spectrum antimicrobials, because if there are a lot of Gram negative
bacteria in the blood stream and you kill them, their endotoxins will be released into the blood
stream, which can be deadly to the patient.
You will be assigned one organism. Use a swab to streak the plate for confluence.
If your plate is a Gram negative organisms, you will be given 12 antibiotic discs that are broad
spectrum. If your plate is a Gram positive organism, you will be given 12 antibiotic discs that are
narrow spectrum. Your instructor will apply the discs to your plate. Place your completed discs in the
sleeve on the counter. Next time, we will measure the zone of inhibition, which is the clear zone
around the disc, which has no bacterial growth. Each laboratory uses the same media and
antibiotic discs, and the size of each disc is always the same. Therefore, this experiment is
standardized and you can use an antibiotic sensitivity table (Kirby-Bauer chart) to evaluate your
results. Because the discs are the same size, you measure from the edge of the disc to the edge of
the clear zone. This is different from the antiseptics experiment, where you measured from one edge
of the clear zone to the other. The zone of inhibition determines the effectiveness of the
antimicrobials ONLY when checked against the Kirby-Bauer chart. Just because one zone is
the largest, it does NOT mean that is the most effective antibiotic, unlike the
antiseptic/disinfectant experiment. Below is an example of an antibiotic sensitivity table. The actual
chart is in your lab manual.
Ampicillin
Amoxicillin
Cephalexin
Cephadroxil
Resistant
<12
<17
<16
<20
Intermediate
13-18
18-20
17-19
21-24
Sensitive
>19
>21
>20
>25
Suppose you measure all the zones of inhibition on your plate, and the largest one measures
20 mm. Is that the antibiotic that is most effective? NO! We cannot answer that question until
we check the chart. According to this sample chart, if the zone of inhibition for your
Cephadroxil disc was 20 mm, the organism is resistant to that antibiotic, and you should
NOT prescribe Cephadroxil to that patient. The zone for Cephadroxil needs to be 25 mm or
more to be effective. If the zone of inhibition for your Ampicillin disc was 19 mm, check the
table and see that the organism IS sensitive to Ampicillin, so that is the antibiotic that you
give the patient. Therefore, the antibiotic with the largest zone of inhibition is NOT
necessarily the most effective one, unlike the antiseptic/disinfectant experiment.
Intermediate sensitivity implies clinical efficacy only in body sites where the drugs are
physiologically concentrated (e.g. quinolones and beta-lactams in urine) or when a higher
than normal dosage of a drug can be used. Choosing an antibiotic in the “sensitive” category
is the best choice. However, if the patient is allergic to that medicine, choosing TWO
antibiotics in the “intermediate” category is the next best option, because they give a
synergistic effect. One might inhibit protein synthesis, while the other interferes with the cell
wall structure. Alone, they cannot kill the organism well, but together they are effective. The
first treatment option for the patient is an antibiotic that is in the sensitive category. If
that is not possible (because there are no such drugs for that organism, or the patient is
allergic to the drug), the second treatment option is to give two antibiotics that are in
the intermediate category (combination therapy), because they have a synergistic effect.
TEMPERATURE REQUIREMENTS OF BACTERIA
Sometimes we incubate plates in a heater or cooler, and sometimes we leave them in room
temperature. Why? Organisms have different temperature requirements. Human
pathogenic organisms prefer our body temperature (37 °C). Non-pathogenic organisms
might prefer room temperature (25 °C). The type of pathogenic bacteria that have flagella
for motility often exist in two different forms: they have a flagella when they are outside of a
human body, and they lose their flagella when they enter the body. Remember, when we
are testing for motility of an organism, we have to incubate at room temperature, or they
will lose their flagella and test as non-motile. Remember our Serratia organism from the red,
white, and blue colony experiment? It only makes the enzyme that turns red at 25 °C, so it
produces red colonies only at room temperature. At our body temperature (38°C) it’s
enzyme is denatured, so it does not turn red; it just stays white. Organisms have various
temperature requirements in order to have maximum enzyme activity.
Psychrophiles: like freezing cold temperatures
Mesophiles: prefer anything from room temperature to body temperature (20-50 °C).
Thermophiles like it quite hot (50-80 °C)
Hyperthermophiles: like extreme heat, such as found in volcanoes.
Most human pathogenic bacteria are mesophiles. We will grow cultures of bacteria at
different temperatures. Next lab time, we will record their transmission readings in the
spectrophotometer and calculate their optical density. Those with the highest OD are the
ones that grew the best at that temperature.
HAND SCRUBBING EXPERIMENT
A nosocomial infection is one that is acquired after a patient is admitted to a hospital.
Nosocomial infections can be avoided by proper hand washing between patients. We will
perform a hand washing experiment and use the wash water to inoculate a series of plates
which are made with Brain Heart infusion agar. It is melted already and in the water bath.
Transient organisms are those which are on our hands for a short while. We pick these up
by touching objects, etc. Resident organisms are those which stay on us. Our resident
organisms do not cause us disease unless there is a break in the skin. However, your resident
organisms might be harmful to another person. Washing your hands with plain water rinses
off a few organisms. Washing with soap and water removes many more organisms.
Additionally, washing for a short time will remove the transient organisms. You have to
wash longer to remove the resident organisms.
We need two volunteers: one to be the doctor, scrubbing their hands, and the other to be the
assistant to the doctor. The doctor will wash his/her hands with the sterile scrub brush in
basin A, rinse, then dunk his/her hands back into basin A. The doctor will then use the same
brush to scrub with soap, and rinse in the sink, and dunk their hands into basin B to collect
the bacteria after the soap and water scrub. Then the doctor will get a new sterile brush and
wash in plain water and dunk into basin C, and then scrub with soap and water and dunk into
basin D. Soap and water are again used, and the hands are dunk into basin E.
Basin A contains the organisms washed off after a short time, with plain water. It will contain
a few transient organisms. Basin B contains the rest of the transient organisms, which were
washed off with soap and water, and after a longer period of scrubbing. That basin will
contain many organisms. Basin C contains only a few of the resident organisms, since only
plain water was removed. The organisms are residents instead of transient because the scrub
time has been longer. Basin D was a long scrub time, and with soap and water, so many of
the resident organisms will have washed off into this basin. Basin E is also soap and water,
and is the longest scrub time, but since most of the resident organisms have been washed off,
there will not be many resident organisms left in that basin. If you graph the results of the
colony counts for each bucket, which two basins will have the highest points on the graph,
and what do these points represent?
Answer: Basin B (soap and water, short scrub time, removes transient organisms).
Basin D (soap and water, long scrub time, removes resident organisms).
Only one doctor (hand scrubber) and one assistant for the entire class will be used to obtain
the inoculated basins. When they are done, the rest of the class will be divided equally into
five groups, each one assigned to make culture plate from one basin A-E. Each lab group
from A-E will take their assigned basin and pipette out the proper amount to inoculate 6
plates (a duplicate set of each dilution).
Plates 1 and 2: pipette 0.1 ml into the sterile plate, add the melted agar, swirl gently.
Note: the dilution factor for these plates will be 10x that, so DF = 10
Plates 3 and 4: pipette 0.2 ml into the sterile plate, add the melted agar, swirl gently.
Note: the dilution factor for these plates will be 10x that, so DF = 5
Plates 5 and 6: pipette 0.4 ml into the sterile plate, add the melted agar, swirl gently.
Note: the dilution factor for these plates will be 10x that, so DF = 2.5
At the next lab, you will count the colonies on ONE of each of your duplicate plates, record
the results on the board with the rest of the class, and graph the results in your lab manual.
Study Guide for Laboratory Calculations
on Dilutions and Concentrations
A. Dilutions Problems
1. Prepare a serial 2 fold dilution to 1:64
2. Prepare a serial 10 fold dilution to 1:10.000
3. Prepare a serial 5 fold dilution to 1:3125
B. Concentration Problems.
What is the concentration of a suspension that results in the following?
1. 35 colonies on a plate with a dilution factor of 1:164
2. 16 colonies on a plate with a dilution factor of 1:320
3. 237 colonies of a plate with a dilution factor of 1:486
Given a Staph aureus suspension with a concentration of 4.6 X 106 organisms/ml,
answer the following questions.
4. What is the concentration of the mixture of 1 ml of Staph. aureus in 3
ml of TSB? Remember concentrations are designated in
organisms/ml.
5. of 3 ml of Staph in 9 ml of TSB?
6. of 5 ml of Staph in 10 ml of TSB?
7. of 4 ml of Staph in 16 ml of TSB?
Answers
A. Dilution Problems
1. 2 fold dilution to 1:64
2. 10 fold dilution to 1:10,000
3. 5 fold dilution to 1:3125
B. Concentration problems
1. 35 colonies X 164 dilution factor = 5740 org/ml = 5.74 X 10 3 org/ml
2. 16 colonies X 320 dilution factor = 5120 org/ml = 5.12 X 10 3 org/ml
3. 237 colonies X 486 dilution factor = 115,182 org/ml = 1.15 X 10 5 org/ml
4. 4.6 X 106 organisms in 4 ml = 1.15 X 106 org/ml
5. 3ml(4.6 X 106 org/ml) / 12 ml = 1.15 X 106 org/ml
6. 5ml (4.6 X 106 org/ml) / 15ml = 1.53 X 106 org/ml
7. 4ml (4.6 X 106 org/ml) / 20ml = 9.2 X 105 org/ml
Dilution and Concentration Problems
1. Prepare a serial 10-fold dilution to 1: 100,000,000
2. Prepare a serial 2-fold dilution to 1024.
3. Prepare of serial 3-fold dilution to 6561.
The concentration of a suspension of Alcaligenes faecalis is 8.4 X 10 5 org/ml.
Answer the following 2 questions using this information.
4. What is the concentration of a suspension with 3 ml of Alcaligenes plus 5 ml
of TSB?
5. of 8 ml of Alcaligenes plus 10 ml of TSB?
6. What is the concentration of a suspension that grew 21 colonies on a plate
with a dilution of 1: 100?
7. What is the concentration of a suspension that grew 84 colonies on a plate
made with 0.1ml from a culture with a dilution of 1:10,000?
ANSWERS
Additional Conc Dil Homework
1) A sample culture contains 3.45 x 106 cells/mL
a. Draw out your plan to prepare five serial, 1:3 dilutions
2) Below is a list of countable plates that several people obtained. Write the concentration of
each original sample.
Colonies from 1st plate = 330
Concentration of original _________________
Colonies from 2nd plate = 221
Concentration of original _________________
rd
Colonies from 3 plate = 110
Concentration of original _________________
Colonies from 4th plate = 98
Concentration of original _________________
th
Colonies from 5 plate = 42
Concentration of original _________________
3) 4mL is removed from a culture of 4.56 x 105 cells/mL and added to 16mL of TSB. What
is the concentration of the new solution?
4) Use your log table to calculate OD from the following transmission readings.
% Transmittance
45.6%
12.3%
78.9%
67.8%
34.5%
9%
8%
LOG
OD
5) What is the concentration of a suspension that grew 48 colonies on a plate with a dilution
of 1: 100?
6) What is the concentration of a suspension that grew 35 colonies on a plate made with
0.1ml from a culture with a dilution of 1:10,000?
7) Given a culture of 7.6 X 10 4 org/ml, what is the concentration of a suspension with 3 ml
of that organism plus 5 ml of TSB?
8) The original concentration of a sample is 4.85 x 104 org/mL. This sample was diluted to
1:2. What is the concentration in the dilution? ______________________________
What is the concentration of a 1:4 dilution of the original? _______________________
What is the concentration of a 1:8 dilution of the original? _______________________
What is the concentration of a 1:16 dilution of the original? _______________________
9) Perform a 1:3 serial dilution, then transfer 1 mL from each tube to a plate. The following
numbers of colonies were counted in each plate. What was the concentration of the
original sample?
Plate 1 (undiluted) = 2000
Plate 2 = 1000
Plate 3 = 500 colonies
Plate 4 = 180 colonies
Plate 5 = 19 colonies
ANSWER ____________________________________
Micro Lab Unit 3 Notes
PRACTICE DICHOTOMOUS FLOW CHART
Each person in here is uniquely identified by their name (assuming there are not two people with
the same name!). I can call Jim Smith, and only one person will respond. Each organism has a
unique name, too. Their first name is their genus, and their second name is their species. But,
unlike Jim Smith, I cannot just call out the name of a bacterium and expect it to respond.
So let’s say that I have a list of your names but I am not allowed to communicate with you. I need
to be able to pick out Jim Smith and Suzie Slowpoke from this class. What tool would I need? It
would be helpful if I had a list of characteristics that uniquely identified each person in this room.
But if all I did was to look at each list of characteristics, and then look at each person to see if
they match the list, it would be too hard if there were 40,000 people in this room.
Therefore, we need to make this list of characteristics into a dichotomous tree (called a
dichotomous key). That means that for each branch of the tree there will be TWO AND ONLY
TWO CHOICES. In this exercise, each deck will work as one group to make a dichotomous key
that will uniquely identify each member in your group. The first branch of the key might be: Male
or Female? That will divide out the sexes in the group. To further subdivide the males, you could
ask about eye color. Can you ask: “Brown, blue, or green eyes?” NO! That is not dichotomous.
You would have to say “Brown Eyes or Not Brown Eyes”. Then for the branch under “Not
Brown Eyes”, you could ask “Blue Eyes or Green Eyes?”
You could also sort people out by age, height, hair color, hair length (don’t say long or short
because that is subject to various interpretation; say “shoulder length or less vs. more than
shoulder length). Note that you cannot sort by clothing because they might not be wearing that
clothing next time. Decide what kinds of characteristics you want to use in your key.
After you go through each characteristic and sort people out, there should be only one person’s
name at the end of each branch of the tree. There should not be any branches that are not
necessary. When you key is done, I should be able to look at it, then look at any one person, and
go through the tree to find that person’s name.
Work as a group to decide how to make your key, and each person should make their own copy of
the same key. It might look something like the sample key below:
SAMPLE DICHOTOMOUS KEY
BERGEY’S MANUAL
In this unit, you will be given an unknown organism to identify to genus and species. The first
thing you will be doing is a Gram stain to make sure only one organism is present. If you have a
contamination, we need to know about it before we go any further. In that case, you might need to
use selective media to get rid of the contaminating organism, while allowing yours to grow.
Once you have performed the Gram stain, you will fill out a pink slip describing your organism’s
Gram stain, shape, and arrangement, turn this slip in to your instructor to confirm that you are
correct. After your pink slip is confirmed and returned, you can then go to your lab manual,
Exercise 39, p. 268-269. If your organism is Gram positive, use the chart on p. 268. It will then
ask you to decide if you have rods or cocci. Make that determination, then decide if you see
spores or not. Continue to follow the tree until you figure out which GROUP NUMBER your
organism fits into. If you have a Gram negative organism, the chart is on p. 269. Once you have
identified your organism’s group, find the description of that group number in your lab manual,
from pages 268-271. Each description will include a list of several organisms. From that
description, you will be able to identify the GENUS of your organism. The description will also
tell you what volume and section of Bergey’s Manual you need to look at to identify your
SPECIES. Bergey’s Manual is a set of 4 books, but all of the organisms in this class will be in
volumes 1 and 2 only. After you know the genus, go to the right volume and section in the
Bergey’s books. You will see many pages that describe the many species for that genus…look for
the table in that section so you can see at a glance all of the laboratory tests that differentiate each
species. That will tell you which laboratory tests you need to perform to identify which species
you have.
Bergey’s Manual is a collaboration of experts from all over the world. Phylogenetic
classification of bacteria is being worked out by sequencing ribosomal subunits. Even with
its discrepancies, the present edition of Bergey’s Manual is the official classification system
for bacteria. In the edition we used of Bergey’s, organisms are grouped together in sections
based on their phylogenetic relationship, according to their physiology (as demonstrated by
the positive and negative lab tests we will perform). When you have an organism that is
positive for a particular test, and you look it up in Bergey’s Manual, it may state that your
organism sometimes is positive for that test, or that it generally appears in a certain way.
Terms like “generally”, “usually”, and “sometimes” refer to the fact that results may vary
from one isolate to another.
DESCRIPTIVE CHART
Before you are given your unknown organism next lab period, we will go through a practice
exercise. You will be given a descriptive chart with the laboratory test results already filled in. To
see what this descriptive chart looks like, look in your lab manual at Exercise 34, p. 235. In class
today, the instructor will hand out these charts, but they are already filled in for you. Start with the
procedure listed above to identify the group by using your lab manual. Then identify the genus by
using your lab manual. Then go to Bergey’s Manual, find the right table, look at the results of the
lab tests on your descriptive chart, and identify your organism’s species. Write down your genus
and species, and have the instructor check the key to see if you are correct.
Go to page 235 in your lab manual to see the blank descriptive chart. You will need to scan this
and print THREE copies. The first descriptive chart (due date is on your schedule) will be when
you can fill in the LEFT side of the page only. The second descriptive chart (due date is on the
schedule) will be when you can fill in the RIGHT side of the page. It is okay to have incorrect
answers on these charts; you do not lose any points for a wrong answer. However, do not leave
any lines blank or you will lose points. After turning in each chart, your instructor will grade it
and give you the correct answers for your organism so you can proceed with trying to identify
your organism, using the correct data. The third descriptive chart should therefore contain all the
correct answers. Make that chart nice and neat and turn it in along with your final report. It will
serve as the results section of your paper. NOTE: on the back of your third descriptive chart,
make a flow chart of how you identified your organism. You can use same format as the flow
chart in your lab manual to get you started, but continue it with the details that enabled you to
identify your organism to genus and species.
When you get your real organism in the next lab period, you will perform several tests on the first
day, and in the following lab period, you will write the results on the left side of a blank
descriptive chart. Then take your descriptive chart to the instructor, who will check to see if any
of your results are in error before you go farther. Do not leave anything blank on your descriptive
chart, or you will lose one point for each blank line. There are no points taken off for any wrong
answers, just blank lines. Once your instructor has checked to make sure your results are what
they should be for your organism, you can go on to perform the necessary lab tests listed on the
right side of the page of your descriptive chart. When your results are in, you again turn in your
descriptive chart to your instructor. Once you have correctly identified your organism, you will
write a report on that organism. Make sure you are not absent during the next few weeks, because
no one is allowed to read your tests or inoculate media for you. Remember, there is one day
coming up that you will need to come in on the day after the inoculation to read the result.
JOURNAL
While you are performing tests to identify your organisms, you will need to keep a scientific
journal to document each test and result. You will be given a handout to use as your journal. All
of your writing on this document must be in pen, just like an actual scientific journal. You are
not to use white-out for errors. You also cannot scribble out any errors. If you make a
mistake, take your pen and make one single line through the word(s) you don’t want, then you
can add the correct word(s). It is okay for your journal to be messy. NOTE: each student only gets
ONE journal handout. You must bring it to class each lab period so you can see what observations
you need to make and what to record. If you forget to bring it, you must use someone else’s to see
what observations you need, write your answers on regular paper, then go home and transfer your
answers to the journal, and do not forget to bring it again. Note: You must write the date you
performed each test listed in this journal, even though there is no space that says to write
the date. Download UNKNOWN REPORT GUIDELINE document from BB and bring it with
you for the next lab period.
UNKNOWN REPORT GUIDELINE (Handout)
This document shows you how to write your report. The PURPOSE statement is just 1-2
sentences. The purpose is to identify an unknown organism, using a series of tests. The Materials
and Methods section will be the longest part of your report. You need to write paragraphs for each
of these sections after each lab period. The MATERIALS section is just a list of the materials
used during the entire project. That includes media, equipment, stains, reagents, etc. The
METHODS section is in paragraph form. Each paragraph should have a heading. One heading
should be “Maintaining Cultures”, with a paragraph below it describing what you did to maintain
your cultures. Then write another heading regarding another method, with your paragraphs below
it, etc. Note that the methods section should NOT contain any results.
Instead of writing a section for RESULTS, you will just be turning in a nice copy of your third
descriptive chart, after you have recorded all of the data CORRECTLY, plus your journal, plus a
flow chart that shows how you identified your organism. In the DISCUSSION section, talk about
your organism, where it lives, any diseases it causes, and how you were able to come to the
conclusion of what organism it is. Describe that it was positive for particular tests (describe the
tests), and which tests were negative, and how those tests told you the name of the organism.
When discussing your organism, you should also look at your descriptive chart and discuss the
characteristics documented there. Your report will wind up being perhaps 11-13 pages (the
method section is long), so start on the first day you get your organism.
Your unknown report is worth a total of 100 points. You will be graded on three things:
1) Three points for your score on the pink slip for the Gram stain, cell shape, and
arrangements
2) Two descriptive charts
3) Your report, which also includes your third descriptive chart, flow chart, and journal
You will mainly be evaluated by the report. It is okay to not have the right organism because one
of your tests did not come out right, as long as you followed Bergey’s Manual and came up with a
logical conclusion of what your organism should be, based on your lab tests.
For the lab exam, you need to understand what media you used, why it was used, and what the
results mean. You also need to know the reagents used, including the names of indicator dyes in
the media, etc.
START UNKNOWN ID
METHOD OVERVIEW
Obtain your unknown stock, and check it for purity by performing a Gram stain. Then use it to
inoculate a working stock and a reserve stock. Each week, you will check your working stock for
purity by performing another Gram stain before using it to inoculate the media to perform the
tests for that day. Use aseptic technique throughout this month. You will not use the reserve stock
unless your working stock becomes contaminated.
Then determine the morphological characteristics of your unknown organism by performing a
Gram stain, motility stab, capsule stain. If you have a Gram positive rod, you will also need to do
a spore stain.
Next, determine the cultural characteristics of your organism by observing the growth patterns
in broth and on agar slants and plates. Determine the optimal temperature and oxygen
requirements, and determine what type of hemolysis your organism displays.
Next, determine the physiological characteristics of your organism. This will require about 1820 individual tests to find out what enzymes your organism makes, its fermentation pathway, etc.
GETTING STARTED
Select a tube from the rack of unknown organisms, check what ID number it is, and write your
name on the sign-up sheet next to the number of your unknown organism. Then write your name
on the tube. Be careful not to contaminate this tube today. Once you have your unknown ID
number recorded on your journal (lab handout), perform a Gram stain. Since the longest step is
air drying, make 3-4 slides and allow them to air dry at the same time, but only use one to
perform your Gram stain. That way, if your culture is not decolorized properly, you have several
slides ready to go so you can perform another stain quickly. When you observe your organism
under the microscope, check to make sure your culture is pure. Sometimes, the Gram stain
becomes contaminated or your culture may be contaminated. If you see more than one organism,
let your instructor know immediately. If you only see one organism, the next step is to do a
Negative stain with India ink, which is the best way to see the arrangements of the organism.
When you know the shape (rods or cocci?) and arrangement (singles, clusters, or chains?) go to
the front of the lab are pink slips. Take one, and record your results. This exercise is worth 3
points towards your 100 pt. unknown report. On the pink slip, write whether your organism is
Gram positive or negative (1 pt.), whether the organism is a rod or cocci (1 pt.), and whether the
arrangements (1 pt.) are singles, clusters (staphylo), or chains (strepto). Then hand this pink slip
to the instructor, who will tell you what the correct answers should be for your organism. Then
record the correct results in your journal and draw the proper pictures there. You may NOT
inoculate any tubes or plates until after your pink slip has been approved.
Once you have your pink slip approved, use your unknown broth to inoculate 3 broths, 2 slants,
and 2 plates. When you are done with your original unknown broth, return it to the rack where
you got it from. It will be stored in the refrigerator in case you lose your culture during the next
few weeks. One of the slants you inoculate today will be the one you use to inoculate new media
next week. Today is the only time that you will be using your original unknown broth tube.
CREATE A WORKING STOCK AND RESERVE STOCK
Inoculate 2 TSA slants by using a needle. Obtain the inoculum and place the needle in the TSA
slant toward the bottom, and pull a straight line upwards on the surface of the slant. One of these
slants will be labeled “working stock”. Your working stock tube is the one used to obtain
inoculums for other lab tests after today. Your transfer schedule is this: Each WEEK, you will
need to make a new working stock tube so your culture stays young. To make a new working
stock, just use your old working stock to inoculate a new tube. After you see growth in the new
tube the following week, you need to check for purity by performing a Gram stain. Once you
know your new working stock is not contaminated, you can discard the old working stock tube.
That means you need to write dates on these tubes. The second TSA slant you will make today
will be your reserve stock in case your working stock becomes contaminated.
MORPHOLOGICAL CHARACTERISTICS:
Gram stain, size determination, motility, capsule stain, spore stain
SIZE DETERMINATION
If you have a Gram + organism, mix a loopful of it with a loopful of a Gram neg organism whose
size is known. If you have a large organism, pick a large organism to compare it with. If you
have a small organism, pick a small organism. Estimate the size of yours compared to the known.
MOTILITY TEST (positive is E. coli, negative is Klebsiella pneumoniae)
Inoculate a motility stab. Use a needle to obtain the inoculum. Stab the needle into the motility
medium, almost all the way to the bottom, then pull the needle back out in a straight line, backing
the needle out of the same stab line you made going in. Remember, these need to be incubated at
room temperature (25°C). If they are placed at room temperature, the flagella will detach, giving a
false negative result for motile organisms. Also remember that motility media uses TTC as a
terminal electr4on acceptor. If the organism can use it, the media will turn red, meaning the TTC
has been reduced. If there is no red color at all, you will need to do a wet mount or hanging drop
to observe the organism directly to determine if it is motile.
CULTURAL CHARACTERISTICS:
Growth patterns, temperature, hemolysis, and oxygen requirements
GROWTH PATTERNS
Use your working stock and reserve stock to observe the growth patterns of your organisms and
record that information in your journal. The terminology to use is on p. 238 in your lab manual.
When you have recorded the morphology on your reserve stock, hand it back in to the instructor
next week, and it will be kept in the refrigerator. You will not use it except in emergency. Also
use your TSB tubes from your optimum temperature experiment to determine the pattern of
grown in broth (see page 238 of your lab manual).
DETERMINE OPTIMUM TEMPERATURE FOR YOUR ORGANISM
Inoculate one loop-full of your organism into 3 TSB tubes. Label one tube 25 °C, one tube 30 °C,
and one tube 38°C. Make sure your name is on the tube. These tubes will be used to determine the
optimal temperature for your organism. Use the spectrophotometer to calculate their optical
density at the next lab period. The tube with the most growth (highest OD) is the temperature they
prefer. Organisms that grow well in room temperature as well as body temperature might be
opportunistic pathogens. These tubes can also be used to determine their pattern of growth in
broth (see p. 238 of your manual).
HEMOLYSIS TEST (Controls: Beta = Staph aureus; gamma = E. coli, alpha = Strep bovis)
Inoculate a blood agar plate. Streak for isolation again. You will use this plate to observe colony
morphology and hemolysis patterns (see p. 358 in your lab manual). Beta hemolysis means the
organism can completely lyse red blood cells and they digest the hemoglobin (pathogenic
bacteria), so there will be clear areas around the colonies on your plate. Alpha hemolysis
means the bacteria can oxidize the iron in the hemoglobin, which turns the colony green,
with NO clear areas. Gamma hemolysis means the organism is non-hemolytic, so there will
be NO clear areas, and the colony will not be green. At the front center deck, you can see the
Controls (positive and negative results) for blood agar plates, motility stabs, and thio tubes. Now
it is time to determine the oxygen requirements by inoculating a sodium thioglycolate tube.
SODIUM THIOGLYCOLATE TUBES (OXYGEN REQUIREMENT)
This medium has an oxygen gradient, which means that most of the oxygen is at the top of the tube, and the
least amount of oxygen is at the bottom of the tube. To prepare this medium, a reducing agent called
Sodium thioglycolate was added, which removes the free oxygen by chemically binding with it. Therefore,
thioglycolate broth is called a REDUCING MEDIUM. It gets rid of the oxygen. There is also a pink
indicator dye called rasazarin that shows you where the oxygen is. Notice that the pink color is only at
the top of the tube. We have to be careful not to shake the tube, or we will aerate it (add more oxygen). We
need the oxygen gradient to be maintained for a successful test. The results of this test determine what
oxygen requirements your organism has.
Procedure:
1. Hold the thioglycolate tube carefully, taking care to move it gently without shaking,
jiggling, or stirring them (which introduces oxygen into the medium).
2. Label the tube with your name, the date, the organism, and “Thio” for Thioglycolate.
3. Put some of your unknown bacteria on a sterile loop and gently push the loop straight
down to almost the bottom of your tube. Do not touch the bottom as this may ruin the
loop, and do not introduce air by stirring or shaking the tube!
4. Gently pull the loop straight out of the tube and sterilize it.
1. STRICT AEROBES require oxygen to grow. There will only be growth on the
surface of the thio broth tube (pseudomonas and Bacillus megatarium)
2. STRICT ANAEROBES require the absence of all oxygen. There will only be growth
at the butt (bottom) of the tube (clostridium).
3. FACULTATIVE ANAEROBES grow best aerobically but do not require it. Growth
is throughout the tube, but is best at the top and decreases as one descends. (E.coli,
staph aureus)
BEGIN PHYSIOLOGICAL TESTS
STREAK FOR ISOLATION
Inoculate a TSA plate, using the streak for isolation method (draw 4 quadrants on the bottom of
the plate, zig-zag the upper left quadrant, flame the loop, then draw the loop from quadrant 1 (Q1)
into Q2 and zig-zag that second quadrant. Flame the loop, then draw the loop from quadrant Q2
into Q3 and zig-zag that third quadrant. Flame the loop, then draw the loop from quadrant Q3 into
Q4 and zig-zag that final quadrant. You will use this plate to observe colony morphology on a
plate (see p. 240 in your lab manual). At the next lab period, you will also use this plate to
perform the catalase and oxidase tests.
CATALASE TEST (Control: positive = Staph aureus)
Some facultative aerobes have the enzyme called catalase, which breaks down hydrogen
peroxide (H2O2) into harmless water plus oxygen. Having this enzyme protects organisms from
being destroyed by the H2O2 in the lysosomes of a white blood cell. Your instructor will lift the
lid on your agar plate next lab period, and put one drop of H2O2 onto the colony. A positive test
will show the oxygen bubbles rising up from the plate. That means the organism has the enzyme,
so it is catalase +. See p. 249 in your lab manual. NOTE: do not get catalase mixed up with
oxidase. Catalase breaks down into oxygen, but is it not the oxidase test!
OXIDASE TEST (Control: positive = Pseudomonas aeruginosa)
Some aerobes have the enzyme called cytochrome oxidase, which is a molecule that is a
terminal electron acceptor in the electron transport chain. Your instructor or lab technician
will have a piece of paper inside a Petri dish at the side window. On a piece of paper, she will
place one drop of the oxidase reagent Dimethyl-p-phenylene diaminic hydrochloride (this
substance is carcinogenic, so you will not be using it). Then she will use a toothpick to obtain the
organism from your TSA plate, and she will scrape the sample onto the drop of reagent on the
paper. The test should be done in comparison to a positive control, because time is essential in
the development of the test results. Count the number of seconds it takes to turn purple and record
the time in your journal. If purple is observed at any time, it is positive for oxidase. If there is no
color change, it is negative. See p. 249 in your lab manual.
NOTES: Even if you figured out what organism you have, you need to continue to perform the
assigned experiments. Know which tests show what color on a positive test: Brown, Orange or
Red, Blue, Yellow, Diffused black pigment, Pink ring on top, etc. Know what reagents are used,
what substrates in the media are broken down, and what the products are.
CITRATE TEST (Control: positive = Enterobacter aerogenes)
Citrate is the sole carbon source in this medium. If the organism can use citrate as its only
carbon source, the medium will become basic. The medium starts out green and turns blue if it is
a positive test. It may only be blue at the top, which is still positive. Acid = green (negative) and
base = blue (positive). A negative tube will also show no growth. Ingredients in the medium
include
1) Indicator dye is Bromthymol blue, which is green when acids are present
2) Nitrogen source is Ammonium salts instead of peptones in order to test the ability
of an organism to use a single specific carbon source
This is the reaction:
Citrate + NH4  increase in pH, turns the slant blue
LIPASE (control: positive = Staph aureus)
The Spirit Blue media has lipids. If the organism has the enzyme lipase, fatty acids will be released, and pH will
decrease (become acidic). This will precipitate the blue dye. A positive result is a dark blue streak in the center of
the plate where you inoculated it. If lipase if produced, the concentration of the blue will increase where it was
inoculated. Having clearing is NOT a positive test; it should be darker blue.
NOTE: If you have a Gram positive organism, and if it is not a spore former, you need to do an acid-fast stain. If it
is positive, you have a mycobacterium, which is not really a Gram negative organism. Negative stains are done if
there is a question about the arrangement of your organism. You can see their arrangements best with a negative
stain.
STARCH (Control = Bacillus)
This media has starch. Some molecules, such as starch, cannot be taken into a bacterial cell because the
molecules are so large. The organism can only use starch if it has an enzyme, called amylase, which can
hydrolyze (break down) the starch into simple sugars that can be absorbed into the cell. We will flood
the plate with iodine, which reacts with starch and turns it black. If the organism has the enzyme, there will
be no more starch left, so there is nothing for the iodine to react with. Therefore, the presence of amylase
will show up as a halo (area of clearing) around the organism (positive test). If the organism could not
use the starch, the starch forms a complex with iodine to give a black precipitate around the
organism. That means the organism is negative for amylase. NOTE: the black color only lasts
a few minutes, so you have to read the test right away before the color disappears.
GELATIN (Control = Bacillus)
Some organisms produce an enzyme called gelatinase, which breaks down gelatin. If the gelatin is
broken down, it becomes liquefied, and can no longer solidify, even when cooled in the refrigerator.
Gelatin is a protein, so gelatinase is a protease. Gelatin is the only thing making the media solid. If it
remains liquid, even after refrigeration, it is positive. Solid is negative.
IMViC
This stands for a series of tests:
1) Indole
2) Methyl Red
3) Voges-Proskauer
4) Citrate
The small “i” does not stand for anything; it just makes pronunciation easier.
The IMViC tests are used to identify an organism in the coliform group. A coliform is a gram
negative, aerobic or facultative anaerobic rod which produces gas from lactose within 48 hours.
The presence of some coliforms indicates fecal contamination. We will perform the indole test as
part of the SIM media. We performed the citrate test in the Simmon’s Citrate media. Now we
need to perform the MR-VP test to complete the IMViC series.
MR-VP TEST (Methyl Red/Voges-Proskauer)
We do two tests with this medium: The MR test and VP test. We will inoculate one MR-VP tube
today, let the culture grow until the next lab period, and then add 5 drops of Methyl Red to
perform the MR test. In the next lab period, we will inoculate a new MR-VP tube, let the culture
grow, and then add alpha-naphthol and potassium hydroxide reagents to perform the VP test.
We are looking for glucose fermentation. Bacteria convert glucose to pyruvate using different
metabolic pathways. One pathway produces unstable acidic products which quickly convert to
neutral compounds. Another pathway (the butylene glycol pathway) produces neutral end
products, including acetoin and 2,3-butanediol. A third pathway is the mixed acid pathway, which
produces stable acidic end products which remain acidic. If an organism produces a lot of acid
from the fermentation of sugars, it can override the buffer in the test media. If this happens,
the amber media will turn red. MR-VP broth differentiates organisms that are single acid
fermenters from organisms that are mixed acid fermenters because it contains over-riding
buffers that affect organisms that are single acid fermenters. An organism that produces only
one type of acid after sugar fermentation will not produce much acid, so the buffer blocks the
media from changing color. But if the organism produces many different kinds of acids, it
overrides the buffer and causes the color to change.
MR = METHYL RED TEST
Methyl Red is a yellow colored pH indicator which turns red if the organism uses the mixed acid
fermentation pathway, which is that pathway that produces stable acidic end-products. If
positive, the enzyme present is formic hydrogenylase. The acids will overcome the buffers in the
medium and produce an acidic pH. When methyl red is added, it will go from yellow to red,
which is positive for an organism that uses the mixed acid fermentation pathway. (Control: E. coli
= pos; Enterobacter aerogenes = neg)
NOTE: Methyl red differs from Phenol red
Methyl Red: starts off yellow, turns red when acids are present (indicating glucose fermentation)
Used in MR-VP test (the first part of the test) for mixed acid fermentation
Phenol Red: starts off red, turns yellow when acids are present (indicating glucose fermentation)
Used in Urea broth and in the Fermentation broths
VP TEST
The VP test is an indirect method of testing for non-acid end products of glucose
fermentation. It detects organisms that utilize the butylene glycol pathway and produce acetoin.
We cannot test for butylene glycol, but we can test for acetoin. The VP reagents are called
Barritts’s A (alpha-naphthol; 18 drops) and Barrett’s B (potassium hydroxide; KOH, 8
drops). These are added to MR-VP broth that has been inoculated with an organism that uses the
butylene glycol fermentation pathway, the acetoin end product causes a rust or red color
(Gram negatives tend to do this). Therefore, red is a positive result, colorless is negative. Shake
gently after adding the reagents, wait 15 minutes, then shake again. (Control: Enterobacter
aerogenes = pos; E. coli = neg)
UREA BROTH (Control = Proteus vulgaris)
This test checks for the enzyme called urease, which breaks urea down into ammonium and carbon
dioxide (water is not a product of this reaction). The ammonium will increase the pH. The medium has
a pH indicator called phenol red. When pH goes up, it will turn bright pink (positive).
CASEIN TEST (skim milk) (Control = Bacillus)
Some organisms produce an enzyme called caseinase (a protease), which breaks down the protein that
makes milk white. It breaks the protein down into small peptides that can be absorbed into the cell. Do
a heavy streak in the center. Positive is a clearing (halo) around the area of growth of the organism
because the milk is broken down and the white color disappears. Casein is what makes milk look
cheesy when it is left unrefrigerated.
NITRATE REDUCTION TEST (control is E. coli)
If nitrate (NO3) has an oxygen molecule removed, it has been reduced. The new molecule is nitrite (NO2). Nitrite
can also be reduced to nitrogen gas (N2) if it loses oxygen. The reactions look like this:
NO3 (nitrate)  NO2 (nitrite) N2 (nitrogen gas)
If an organism has the enzyme called nitrate reductase, it can reduce nitrate like this:
NO3 (nitrate)  NO2 (nitrite)
Is this enzyme clinically important? Not really. Some of you have brown eyes and some of you do not have brown
eyes. It serves as a way of classifying organisms on a flow chart.
The nitrate broth we started with contains nutrients plus NO3, and it is clear. If is reduced, the tube is still clear, so
how can we tell if NO3 was reduced to NO2? If the organism has nitrate reductase, it will reduce NO3 to NO2 so
there will be no more NO3 present , just NO2. First, we add reagent A to the tube. Reagent A will bind to NO2 ,
forming a complex. However, this complex is clear also, so it does not tell us anything. Then we add Reagent B,
which turns the complex a red color. If you add reagents A + B and the tube turns red, the organism has
nitrate reductase.
However, some organisms with that enzyme reduce NO3 all the way to N2. They take all of the nitrate and reduce it
all the way to nitrogen gas, as seen in this equation:
NO3 (nitrate)  NO2 (nitrite) N2 (nitrogen gas)
In this case, there will be no NO3 or NO2 in the tube, so there is nothing for reagent A to react with, and reagent B
will not turn the tube red, even though the organism has the nitrate reductase enzyme. Therefore, if you add
Reagent A + B, the tube will be clear, yet the organism has the nitrate reductase enzyme. So, although a red color
is a positive test, a colorless tube is NOT a negative test.
When the tube is colorless, there are two possibilities:
1) The organism does not have nitrate reductase, and there is still NO3 in the tube
2) The organism has nitrate reductase, and there is no NO3 or NO2 in the tube.
If a tube is colorless after adding Reagents A + B, we need to test the tube to see if there is NO3 in the
tube. We do this by adding a little zinc powder by scooping some on the flat end of a toothpick and adding
it to the tube. Zinc will react with NO3 if it is present (reduces any residual nitrate to nitrite) and it
will turn red. Zinc is used to confirm a negative test.
Reagent A is sulfanilic acid
Reagent B is alpha naphthalamine
Reagent C is zinc powder
A +B Red is
positive
A +B +C Red
is negative
DECARBOXYLASE BROTHS
(Controls for ornithine: Enterobacter aerogenes = pos; Klebsiella pneumoniae = neg)
This tests for the presence of the enzyme decarboxylase. This test is useful for differentiating the
Enterobacteriaceae. This enzyme removes and digests the acidic carboxyl group (COOH) from
amino acids, plus cleaves off NH3, which will raise the pH. The pH indicator is bromcresal
purple. The media is made to start out slightly acidic (pH 6). Bromcresal purple is yellow when
acids are present and purple when bases are present.
Three tubes are inoculated. Each tube contains glucose plus one amino acid; either lysine,
arginine, or ornithine. The carboxylase reaction requires an anaerobic environment, so each
tube needs to be covered will a layer of sterile mineral oil to prevent air from reaching the culture.
NOTE: PICK THESE TUBES UP FROM THE RACKS ONE AT A TIME AND LABEL THE
TUBE BEFORE YOU PICK UP THE NEXT TUBE. THEY ARE ALL THE SAME COLOR
AND YOU MIGHT GET THEM MIXED UP!
Each decarboxylase enzyme produced by an organism is specific to the amino acid on which it
acts. Therefore, we test the ability of organisms to produce arginine decarboxylase, lysine
decarboxylase, and ornithine decarboxylase using three different but very similar media.
If an organism is able to decarboxylate the amino acid present in the medium, alkaline byproducts
are then produced. Ornithine decarboxylation yields putrescine (named after its putrid smell).
Lysine decarboxylation results in cadaverine (smells like a cadaver). These byproducts are
sufficient to raise the pH of the media so that the broth turns purple (in 48 hours). If you check it
in 24 hours, you might see that it is yellow because it fermented the glucose in the medium, but
that does not mean it is a negative test. You have to check it in 48 hours to allow the
decarboxylase activity to occur. If the pH becomes alkaline because the organism has the
decarboxylase enzyme, the media will turn purple in 48 hours (pos).
DNASE TEST (Control = Serratus marcescens or Staph aureus)
This tests for the presence of the enzyme, DNAse. It contains the indicator dye, Methyl green
complexed with DNA. Digestion of DNA releases the dye, so in otherwise green agar, a clear
halo formed around the growth indicates a positive test.
PHENYLALANINE AGAR SLANT (Control = Proteus vulgaris)
We are looking for the enzyme, phenylalanine deaminase, which removes an NH2 group from
cysteine to produce pyruvic acid, ammonia, and hydrogen sulfide. When 5 drops of ferric
chloride is added to this, it will turn green, indicating a positive test. A negative test stays yellow.
Don’t get this mixed up with the SIM media, where ferrous sulfate turns the media black.
SIM MEDIA
Get one SIM tube and use a needle to stab the media with your organism. Next lab period, we will
add 10 drops of Kovac’s reagent to the tube and check for three things on this one tube.
1) H2S (sulfur) PRODUCTION: Certain bacteria produce H2S from the enzyme cysteine
desulfurase. When the H2S reacts with ferrous sulfate in the medium, a dark precipitate
of iron sulfide is produced and the media will turn black (positive for H2S production).
(Control = Proteus vulgaris). Don’t get this mixed up with the phenylalanine test, where
the addition of ferric chloride turns the media green.
2) INDOLE PRODUCTION: Tryptophanase breaks tryptophan (an amino acid) down
into indole, pyruvic acid, and ammonia. If tryptophnase is present, the indole end
product reacts with the reagent we will add next time (10 drops of Kovac’s reagent). If a
red ring forms at the top of the tube, it is positive for indole, so the organism makes
tryptophanase. (Control = E. coli).
3) Motility: You have already done a motility test, but this media will show you again if your
organism is motile. The red dye is not in this media, so visualization is harder.
FERMENTATION BROTHS (Control = E. coli is AG)
NOTE: PICK THESE TUBES UP FROM THE RACKS ONE AT A TIME AND LABEL THE
TUBE BEFORE YOU PICK UP THE NEXT TUBE. THEY ARE ALL THE SAME COLOR
AND YOU MIGHT GET THEM MIXED UP!
We are looking for fermentation with acid (A) or acid + gas (AG). If there is fermentation, it will
be yellow. If there is gas, the inverted miniature tube inside the media will fill with a gas bubble.
If there is no fermentation, it is red, so record it as no change (NC) or Alk (protein digestion).
The medium has a Durham tube (a miniature tube that is inverted on the inside of the test tube).
If gas is produced, it will form a bubble inside the inverted tube. It also has phenol red as an
indicator. Phenol red turns yellow if acid is present, and red if bases are present.
Inoculate one each of the following tubes: glucose, lactose, mannitol, sucrose, and trehalose.
After 24 hours, if the inoculated medium is yellow, it fermented the sugar in that tube. It may or
may not have produced gas. Gas is produced during sugar fermentation, so when gas is present,
fermentation is present as well, but not all organisms ferment with gas. If it is yellow, record it as
(A). If it has gas in the Durham tube (a bubble that take up 10% of the tube, not a little bubble),
record it as (AG). If it did not turn yellow (stays red), you have to look at it again in another 24
hours. After 48 hours, if the media is still red, the organism is negative for fermentation of that
sugar.
These tubes must be read in 24 hours, because in 48 hours, any change in color will revert to
the original color.
This is what happens:
Some organisms that ferment sugars can also digest proteins. When these organisms begin to
ferment a sugar, the media becomes acidic (yellow in 24 hours), which enables them to begin
digestion of the proteins which are in the media. When proteins are digested, the media becomes
alkaline, and the media will turn back to red. If you want to know if it fermented the sugar, you
need to read the tube in 24 hours.
Suppose a student did not observe their tube right away, and then they see that it is red but
it has gas. Since the gas is present, that indicates that it probably fermented the glucose
(turned yellow at 24 hours, but he missed it), and then the organism proceeded to digest the
protein, turning the media alkaline (back to red again). That would explain why it was red,
but has gas (gas is produced during the fermentation process).
OXIDATION-FERMENTATION (O-F) TEST FOR GLUCOSE
We are looking for the ability to ferment or oxidize glucose. The pH indicator is Bromthymol
blue, which is yellow when acid is present. You will STAB two O-F tubes of glucose. One tube
will need a layer of sterile oil to create an anaerobic environment so we can check for
fermentation. The other tube will not have oil, so we can have an aerobic environment to check
for oxidation. Next time, you will see if it turns yellow. If it is yellow, record it as “A “ (acid
present). If there is gas in the tube, also record “G”. If there was no change (stayed green), write
“NC”.
With oil
AG
NC
NC
Without oil
AG
A
NC
Results
Ferments glucose
Oxidative
neither
Control
E. coli
Pseudomonas aeruginosa
Alcaligenes faecalis
NOTE: According to your lab manual flow chart, know what the first test is after a person
has identified if their organism is Gram positive (do a spore stain) or Gram negative (use a
thioglycollate broth to determine oxygen requirements).
Note: We are boiling down all you tubes today. DO NOT THROW AWAY THE DURHAM
TUBES! To collect them, pour out your fermentation tubes through the strainer provided at the
trash can. That will cause the Durham tubes to fall into the strainer. Do not clean them; just leave
them in the strainer. Many students break test tubes while cleaning up these tests. This happens
because they are trying to handle too many tubes at once. Be careful!
NOTE: Be able to match enzymes to their tests, substrates, reagents, and controls. You should
make a table to study from. In one column, put the name of the test. In the other columns, put the
substrates, products, enzymes, reagents, etc., and what a positive and negative result would look
like.
END OF MATERIAL FOR IDENTIFICATION OF UNKNOWN BACTERIUM
EXAMINATION FOR COLIFORMS IN WATER
Pubic drinking water supplies must be monitored for the presence of coliforms (bacteria that are
found in our bowels). Coliforms are Gram negative facultative rods that ferment lactose,
producing acid and gas. If ingested, they cause serious illness. If they are present in drinking
water, the CDC will shut down the facility until the problem is cleared up. The test for coliforms
in water has three phases:
1) Presumptive Test
2) Confirmation Test
3) Completed Test
Presumptive Test (work in pairs)
Place 10 ml of water into each of three tubes of double-strength lactose.
Place 1 ml of water into each of three tubes of single-strength lactose.
Place 0.1 ml of water into each of three tubes of single-strength lactose.
NOTE: this is not a serial dilution. The same amount is in each tube.
These need to incubate for 18 hours, then we will take them out of incubation and place them in
the refrigerator. At the next lab period, and count the number of tubes in each set that have gas.
Then we will use a table (Ex 45, p. 314) to calculate the Most Probable Number (MPN). Then we
will go on to the confirmation test, because right now, all we know is that we have bacteria that
ferment lactose. They may not be Gram negative rods.
Most Probable Number
Suppose you had 2 tubes with gas in the first and last set, and 3 with gas in the second set.
Your MPN would be 43. That is the number of organisms per 100 ml.
No. of Tubes Positive in MPN in the
first
set
middle
set
last
set
inoculum of the
middle set of
tubes
2
3
1
36
2
3
2
43
2
3
3
53
3
0
0
23
3
0
1
39
3
0
2
64
3
0
3
95
3
1
0
43
To use the table, write down the number of
positive tubes in the first set, the number of
positive tubes in the second set, and the
number of positive tubes in the third set.
Then find that number combination on the
table and record the MPN for that
combination. This is just a small section of
the table.
NOTE: The table in the lab manual does not contain the combination 3-3-3, so if that is the
combination you get, use the combination 3-3-2 in your lab manual.
Confirmation Test
We will use two media which are selective and differential. Each group of two people will work
with one of each plate. Use the water sample to streak for isolation on the plates.
EMB is selective because it has eosin and methylene blue, which only grow Gram negative
organisms. It is differential because it shows lactose fermentation. Colonies with a black
center is a positive confirmation test, since only coliforms will form colonies with black nuclei
on EMB agar. When they show a green metallic sheen, that is positive for lactose fermentation.
MacConkey’s agar is selective because it has crystal violet and bile salts, which only grow
Gram negative organisms. It is differential because it shows lactose fermentation by turning a
pink color. Pink is positive for lactose fermentation.
Whether the confirmation tests were positive or not, we go on to the completed test, because right
now, all we know is that we have Gram negative bacteria that ferment lactose. The may not be
rods.
Completed Test
Inoculate a lactose broth and a TSA slant. Next time we will do a Gram stain. Take a needle;
touch it to one of the colonies and do a Gram stain to check for morphology. If we have a Gram
negative rod that we know is a lactose fermenter, which means we either have Enterobacter (not a
coliform) or E. coli (which is a coliform). Now we have to do an IMViC test to see which one we
have.
E. coli
Enterobacter
Indole
+
-
Methyl Red
+
-
V-P
+
Citrate
+
P-Glo
You should have downloaded the PGlo handout. Work in groups of four.
The pGLO plasmid is an engineered plasmid used in biotechnology as a vector for creating
genetically modified organisms. The plasmid contains several reporter genes, most notably for the
green fluorescent protein (GFP) and the ampicillin resistance gene. GFP was isolated from the
jelly fish Aequorea victoria. Because it shares a bidirectional promoter with a gene for
metabolizing arabinose, the GFP gene is only expressed in the presence of arabinose, which
makes the transgenic organism fluoresce under UV light. GFP can be induced in bacteria
containing the pGLO plasmid by growing them on +arabinose plates.
pGLO is made up of three genes that are joined together using recombinant DNA technology.
They are as follows:
-Bla, which codes for the enzyme beta-lactamase giving the transformed bacteria resistance to the
beta-lactam family of antibiotics (such as of the penicillin family)
-araC, a promoter region that regulates the expression of GFP (specifically, the GFP gene will be
expressed only in the presence of arabinose)
-GFP, the green fluorescent protein
P-Glo is a transformation exercise. We briefly talked about plasmids and transformation in
lecture. Some organisms have the ability to take up DNA from their environment. We will use E.
coli and force it to take up DNA in the form of a plasmid. In E. coli, there is a promoter region for
ampicillin resistance. Another promoter region contains the gene for the sugar, arabinose
(inducible operon). E. coli requires arabinose to turn the gene off.
We have a plasmid which contains the gene from a jellyfish that allows the organism to glow
under florescent light. We are going to introduce the gene into the E. coli, and grow them in plates
(LB instead of TSA). Two plates will have E. coli which are ampicillin resistant, two plates will
have E. coli which are not ampicillin resistant. One of each of those plates will have E. coli which
has had the plasmid inserted, the other will not. One of the other two plates (positive; contains the
plasmid) will contain arabinose, the other will not. We will be incubating in the presence of
ampicillin, and that will select for those organisms which can take up the plasmid.
We will also be incubating for the presence of the sugar, arabinose, because it is required to turn
the gene on. If there is no gene for ampicillin resistance or arabinose, it should not be able to
glow.
Take one colony and place it in one of each of the pink and blue tubes.
The technician will put plasmid in the pink tube. Put both of the tubes into ice for 10 minutes to
force the transformation. Each tube also contains calcium chloride, which pokes holes in the outer
membrane, allowing the DNA to gain access to the inside of the cell. Now we have to shock the
cell to cause the DNA to be taken up. To do this, put them into the water bath at 57 degrees for 50
seconds, then back on ice for 2 minutes. Then collect your tubes and add 250 µl of LB broth to
both tubes and incubate at room temperature for 10 mins. This allows for replication of the cell
and incorporation of the plasmid. Then pipette 100 µl from the blue tube to the two negative
plates (contain no plasmid; one has arabinose and one does not), and pipette 100 µl from the pink
tube to the two positive plates (contains plasmid; one has arabinose and one does not). Next lab,
we will expose them to UV light to excite the proteins and see which ones show green
fluorescence. The only plate that should glow is the one with the plasmid plus arabinose.
Plates
LB/amp positive (has plasmid)
LB/amp/arabinose; positive
LB/amp negative (before transformation, they have no amp resistance. Should not glow)
LB negative (just a media like TSA; will grow, but no glow)
The promoter region (can turn off and on; is an operon. It is turned off and on by arabinose).
The non-transformed cells do not have the ampicillin resistance gene.
Negative
Positive
ELISA TEST
Enzyme-linked immunosorbent assay (ELISA) is a biochemical technique used mainly in
immunology to detect the presence of an antigen in a sample. An unknown amount of antigen is
affixed to a surface, and then a primary antibody is applied over the surface so that it can bind to
the antigen. This antibody is linked to an enzyme, and, in the final step, a substance containing the
enzyme's substrate is added. The subsequent reaction produces a detectable signal, most
commonly a color change in the substrate.
Enzyme: horseradish peroxidase
Secondary antibody
Either antigen or primary antibody
ELISA tests could also use an antibody instead of the antigen. In this case, there will be two sets
of antibodies, so we call them primary and secondary antibodies. The primary antibodies will be
attached to the plastic plate, and then the secondary antibodies will attach to the primary
antibodies. The secondary antibodies will then be conjugated to the enzyme, horseradish
peroxidase, which will create a color change when a substrate is added. An ELISA test can tell us
whether or not particular antigens or antibodies are present in the sample (qualitative). However,
we cannot measure how many antigens or antibodies are present (quantitative) unless we perform
a serial dilution.
For this exercise, you will receive a fluid sample that you pretend is from your body. It is labeled
with a number; write that down. One of these samples is positive for an antigen (we are
pretending it is positive for HIV). Then you will go around the class (between decks too!) and
pretend to transfer body fluids. You put your fluid in someone else’s tube, mix it, and take half of
it back. After the whole class has donated once, then go around and donate a second time. Keep
track of who you donated to, and who you received donations from. You can donate only twice
but you can be the recipient as many times as you want. After performing the ELISA test, if your
sample is positive, track down where you might have gotten it from. Try to figure out who had the
positive sample in the beginning.
Procedure
The 96 well plates have letters down the left side and numbers across the top. ELISA uses
positive and negative controls. The first three wells on the top row are used for positive controls,
the next three for negative controls. The patient’s fluid samples are done in triplicate, so each
student will take up three wells. Decide with the other people in your deck as to who will use
which of the leftover wells in the plate; write your well numbers down on a table. ELISA’s are
run in triplicate for several reasons: to circumvent the possible false negatives, to circumvent the
possible false positives, and to increase statistical significance of the reactions.
Once everyone in your deck has added their fluid to their assigned wells, let them incubate for 5
minutes and wash it with a buffer that will wash out any unbound antigen. Then put in a primary
antibody (since we are pretending the antigen is HIV, the primary antibody would have to be an
anti-HIV antibody). Let it incubate for 5 minutes to allow it to bind to the antigen if the antigen is
present. Wash again with wash buffer, which will wash away any unbound antigen. Then add a
secondary antibody. These are anti-human antibodies; antibodies against human antibodies. These
secondary antibodies also have a horseradish peroxidase (HRP) enzyme attached to them. Allow
the tray to incubate another 5 minutes, then wash with the buffer. Now add the substrate, which
will bind to the HRP if HRP is present. When HRP comes in contact with the substrate, the color
changes to blue. If the blue color appears, it means that the substrate found HRP to bind to. If
HRP is present, the secondary antibodies must be present. If the secondary antibodies are present,
that means the primary antibodies are present. If the primary antibodies are present, that means
the antigen is present, so a color change is positive for the antigen (which we are pretending is
HIV).
END OF MATERIAL FOR UNIT THREE
APPENDIX
Log Tables and Logarithms
The log table in your lab manual is an abbreviated table. There are books of logs but
this abbreviated table is adequate for our purposes. First, let's examine the table. A
small portion of the table is below. There is a left hand column of numbers starting with
10 and ranging to 100. Across the top of the table are the numbers 0 through 9. This
table allows you to determine the log of numbers with up to 3 digits using both the left
hand column and the top lane. The location of the decimal is up to the user. For
example, you could use the table below to locate the number 104 or 10.4 or 1.04.
…
N
0
1
2
3
4
5
10
0000
0043
0068
0128
0170
0212
11
0414
0453
0492
0531
0569
0607
12
0792
0828
0864
0899
0934
0969
13
1139
1173
1206
1239
1271
1303
14
1461
1492
1523
1553
1584
1614
Now let's examine logarithms (logs). A log has two parts; a character and a mantissa.
The characteristic is a number that is calculated and the mantissa is read off the log
table. For instance in this log 1.0531, the characteristic is the number preceding the
mantissa by a decimal i.e. the character is 1 and the mantissa is 0531. The
characteristic is calculated by taking the total number of whole numbers of the figure that
you are finding a log for and subtracting one. For example; the characteristic for 104 is
2, the characteristic for 10.4 is 1 and the characteristic for 1.04 is 0. The mantissa is
read off the table, so for 104 the mantissa is 0170. However, the mantissa for 10.4 and
1.04 are also 0170; the difference in the log for all three numbers is the character.
Examples:
Number
124
12.4
1.24
characteristic
2
1
0
mantissa
0934
0934
0934
logarithm
2.0934
1.0934
0.0934
To determine the log for a number less than 10, use the number with two zeros following;
the log for 5 would be read off the table at 500. The characteristic is 0 and the mantissa
is 6990, hence the log is 0.6990.