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Introduction to Clinical Microbiology
2nd Edition
Microbiology Lab – Fall 2009
Modified by J. Volpe
Table of Contents
Table of Contents ............................................................................................................................ 0
Course Objectives and Information: ............................................................................................... 1
Microbiology Laboratory Rules:..................................................................................................... 2
Microscope Techniques .................................................................................................................. 3
Plating Bacteria ............................................................................................................................... 6
Sterile Technique, Spreading and Streaking Plates. ....................................................................... 9
Gram Stain .................................................................................................................................... 12
Carbohydrate Fermentation (Phenol Red Broth) .......................................................................... 15
Catalase Activity ........................................................................................................................... 17
Obtaining a Pure Culture .............................................................................................................. 19
Acid Fast Stain .............................................................................................................................. 21
Environmental Affects on Bacterial Growth ................................................................................ 24
Antiseptics and Antibiotics ........................................................................................................... 27
The Enterotube .............................................................................................................................. 30
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Course Objectives and Information:
1. Demonstrate basic microbiology lab skills
2. Demonstrate lab techniques including:
a. Documenting accurate experimental lab observations
b. Evaluating lab observations
c. Write comprehensive lab reports
d. Design and verify lab protocols
3. List the responsibilities and duties of a professional microbiology lab
Student expectations:
1. Attend all labs.
a. If you cannot attend your section of the lab: attending the other section is preferred
with prior approval.
b. Labs build on each other; it is crucial to keep up. If you miss a lab you are responsible
for knowing the techniques in that lab because you will use them in other labs.
2. Complete Lab Reports.
a. Lab reports are to be typed and turned in the following lab.
b. Lab techniques are to be completed with a lab partner. Each partner is to complete
their OWN lab report.
3. ACADEMIC HONESTY is expected! Plagiarism and academic dishonesty will result in
a zero for the assignment for the first offense, an “F” for the second.
Lab Report Requirements:
-must include you AND your lab partners name on the report
-Include:
1. Introduction - describe procedure
2. Observations – document detailed observations
3. Exercises – one must answer all questions associated with each lab
4. Conclusion:
i) describe objective(s) you learned
ii) what were the difficulties in the lab technique
Lab grade consists of:
See your syllabus. There will be lab questions on exams and at least one lab essay question per
exam, all assigned information from the in lab manual is testable material.
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Microbiology Laboratory Rules:
General Rules
1. Be courteous to your lab mates, class mates, and instructor. Help each other; you’ll be your best resources.
2. You are expected to have read lab materials before coming to class, lecture materials before an exam.
3. No talking, phone or computer use during exams. In lecture and exams, turn phones off or to vibrate.
4. If you have a problem, speak to your instructor.
Lab Rules
1. Report spills and accidents to your instructor.
2. Wash your workbench top with disinfectant before and after and your hands after each lab. This is good
lab procedure and P1 rules.
3. No food or drink in the lab. No smoking, chewing gum, or chew is allowed. You must wear closed toed
shoes in lab.
4. Your gear, coats, backpacks, etc should be in the back of the lab and not at your workbench. Books,
notebooks, pencils, may be at your bench.
5. Long hair has to be tied back for fire safety reasons. No exceptions when a Bunsen burner is used
anywhere in the lab!
6. Turn off your Bunsen burner when not in use.
7. Assume every organism is hazardous. Do not put fingers, pens, or other objects in your mouth while in the
lab. Gloves are to be worn when handling known pathogens.
8. Cultures that you are finished with need to be autoclaved and thrown away.
9.
Dispose of all materials properly. All cultures or items that have touched a culture are assumed to be
hazardous and must be disposed of properly in a hazardous waste bag. If unsure, ask your instructor.
a. Any instruments that come in contact with bacteria must be sterilized.
b. Inoculation loops are to be flame sterilized before and after each use.
c. Plates must be autoclaved before thrown away. Place used plates in a red biohazard bag in back of
class. Plates are autoclaved in this bag and then thrown away in garbage.
d. Glass tubes and pipettes need to be autoclaved and then washed before reuse.
e. Slides are to be placed in a beaker with disinfectant; they will be washed and reused. Cover slips
are to be discarded as broken glass or if plastic, the autoclave bag.
10. No cultures can leave the lab for any reason.
11. All supplies should be returned to the back of the class where you picked them up from at the beginning of
lab. Lids must be securely fastened.
12. Properly label all tubes and plates w/ your name, the date, and the name of the culture (or the experiment
name).
13. Handle all tubes and plates by gripping the tube or plate, not the lid! You also will need to wash some
glassware.
14. The slide boxes stick. When opening, put a slide box on a flat surfce & carefully pry open the lid.
Otherwise all the slides may fly out and break on the floor.
15. Stains used will stain clothes, lab benches, skin. Contain mess by covering lab benches with paper towel or
lab bench cover, wear gloves, and work over a sink or large beaker.
16. Clean, carry, and maintain your microscope in the proper fashion.
17. Immersion oil is to be cleaned off the oil immersion lens after each use; this is to be done with a piece of
lens paper. DO NOT USE A KIMWIPE or PAPERTOWEL!!
18. Do not deviate from a taught technique w/o permission from the instructor. You will be taught techniques
to minimize contamination of yourself and the surrounding environment as well as sterile techniques.
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Microscope Techniques
Objective:
1. Identify components of the light microscope and demonstrate their functions
2. Identify components and demonstrate function of fluorescence microscopy and
electron microscopy
Resources:
Your textbook
Supplemented by lecture and lab notes
Supplies:
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light microscope
slide
water bottle
lens paper
oil immersion oil
ethanol
kimwipe
scissors
Lab procedure:
1. Cut a lower case “e” from the newspaper.
2. Take a clean slide. If slide is not clean wipe with a kimwipe and a drop of ethanol.
3. Mount the “e” to the slide by placing a drop of water, from a water bottle, on the slide
and place “e” on top. Make sure that the “e” is not moving around. Note which way
the “e” is facing.
4. Starting with the lowest setting focus the microscope and work to higher settings until
the entire “e” is visible. Draw the “e” in lab report viewed under the microscope and
in plain sight.
NOTE: be sure the provide magnification below and label
5. Take a fixed slide from front of class. Work with the microscope to get a clear view
of the culture. Try moving all the functional components of the microscope to see
what makes a clear image.
The microscope will be adjusted differently for everyone’s eyes.
a) All microscopes in the lab have two eye pieces; you should use both
of them. It is common to want to close one eye when starting to use a
microscope, concentrate on using both; adjust the width to make the
lenses fit the diameter of YOUR face.
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b) Change the light intensity to get the clearest view and best colour
illumination.
c) The diaphragm can concentrate light.
6. Starting with lowest magnification, work up until using oil immersion lens. Draw in
lab report.
7. Repeat this process with 3 fixed slides. Drawing each in your lab report.
8. After using oil immersion the lens MUST be cleaned by wiping off immersion oil
with a lens paper. DO NOT USE A KIMWIPE!!!!
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Exercises:
1. Fill in blanks in on microscope image. For components that are adjusted, write a brief
description of what happened to the image when you adjusted it.
2. What did you notice about the plain view “e” and the microscope image of the “e”?
3. What are the lowest magnification and the highest magnification on the microscopes
in lab?
4. When adjusting the microscope from 400X to 1000X you should use the course focus
adjustment, True or False?
5. To get a dark field how would you adjust the microscope?
6. When is it best to use dark field microscopy?
7. Describe a possible usage of fluorescence microscopy.
8. EM is particularly useful is imaging of what type of pathogen? Why?
9. When putting microscope away; in what position (magnification/stage/cord/cover)
should the microscope be in?
10. Explain the importance of using lens paper.
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Plating Bacteria
Objective:
1. List the general ingredients and the major differences between agar, media, and broth.
2. List when you would use agar, media, and broth.
3. Demonstrate pouring agar on a plate / tube or slants using sterile techniques.
Supplies needed:
□ LB or TSA
□ E. Flask
□ Weigh boats
□ Spatula
□ dH20
□ plates
□ tubes
□ tube racks
□ stir bar/rod
□ gloves
□ graduated cylinders
□ Bunsen burner/striker
Procedure for making agar plates:
1. Calculate how much agar you will need to make 250ml of each. You are only
responsible to make one type of agar, assigned in class.
a. LB: 35g/L
b. TSA: 40g/L
2. Weigh agar: using a spatula scoop dehydrated agar out of container onto weigh boat
on scale.
***Do not take agar that has been out of the container and put it back into the
container, this is not done to keep the stock container non contaminated. This rule
should be followed when measuring anything from the lab stock.
3. Measure 250mL H20; half of the class will make LB and half will make TSA.
Assigned day of lab.
4. Combine agar with H20 in an Erlenmeyer flask.
***Best if you put agar into the flask first and then H2O because the agar will stick to
the sides of the flask where the water is, making it more difficult to mix all the agar
into solution.
5. Boil to get agar into solution. Place flask on hot plate starting with heat at about 5 on
a 10 scale to start. Every hot plate is slightly different so it is best to start at a lower
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temperature and increase. When available use a stir bar to mix. In this lab we will use
a stir rod to make sure agar is mixed into solution.
***Do not leave hot plate unattended when turned on.
6. Autoclave: 15min 121oC
a. we will autoclave all at once so wait for the rest of the class if completed
early.
b. Autoclave demonstration will be done.
7. Allow to cool to 56oC
a. This can be done using a water bath or by leaving at room temperature.
8. Pouring:
a. Plates: lift lid at an angle above plate with less dominant hand. With dominant
hand pour agar solution to cover plate
b. Tubes: fill tube 1/3 full with agar solution
c. Slants: fill tube 1/3 full with agar solution. Prop test tube rack so that agar
hardens at a slant
9. Allow to cool and harden.
10. Invert plates and stack.
11. Place lids on tubes.
a. Tubes can be used as tubes or agar can be heated up again and poured into
plates.
12. Refrigerate 4oC for storage
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Exercises:
1. Write step-by-step protocol for making an agar plate. Include details.
2. How much dehydrated agar (LB and TSA) was needed to make 250ml of agar? Show
calculation.
3. When would you add antibiotics, if you wanted an ampicillin slant?
4. What is the advantage of a plate, tube, slant?
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Sterile Technique, Spreading and Streaking Plates.
Objective:
1.
2.
3.
4.
5.
List how to control contamination
List why contamination control is important
Perform techniques to prevent contamination
Perform spreading and streaking techniques
List proper way to clean up after a microbiology lab and why its important
Resources:
Textbook chapters.
Supplemented by lecture and lab notes
Supplies:
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4 different broth bacteria
dropper/bulb
beaker
L rod
Inoculation loop
Ethanol
Sharpies
Bunsen burner/striker
Plates (from 2a)
Procedure:
Spreading:
1. Pipet 1ml bacteria on agar: You will use your plates you made in 2a, broth
bacteria cultures will be provided at front of class. For this lab quantity is not
important therefore we will use a dropper and bulb to transfer bacteria. After
use both should be placed in 10% Lysol solution.
2. Dip L-rod in ethanol and flame L-rod. Flaming is a way of sterilizing
instruments; the flame of the Bunsen burner kills the bacteria that are on the
instrument. This is the same sequence used to flame an inoculation loop or
needle.
NOTE: Make sure to hold rod at an angle so that ethanol does not run
towards your hand.
3. Allow L rod to cool. If you do not allow your L rod to cool the heat can kill
the bacteria on the plate.
4. Spread bacteria. Using L rod distribute bacteria evenly across surface of agar.
5. put in incubator for 24-48 hours
6. Draw and record you observations.
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Plate Streaking (Colony Streaking -Quadrant Method):
1. On the bottom of an agar plate divide the plate into 4 quadrants. Write your
initials and date. Label each quadrant with bacteria name that you will be
streaking that quadrant with. Leaving one quadrant labeled control.
2. flame sterilize inoculation loop. Same procedure as L rod but your can cool
loop by placing loop in on an agar plate. The loop will cause a ring in the agar
plate.
3. streak one quadrant of plate with one bacteria.
4. flame and repeat with next bacteria
5. leave 1 quadrant not streaked as a control
6. put in incubator for 24-48 hours
7. Draw and record observations
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Exercises:
1. Why is it important to use sterile technique?
2. Media, broth and agar are sterilized using different techniques. What are they and
why are they different?
3. Give 2 reasons why wearing gloves in lab is important.
4. Name 5 body sites that are normally sterile and 5 body sites that are not. Why is it
important to know this information?
5. What should you do when finished with a plate, tube or instrument that has come in
contact with pathogen?
6. What would be the function of adding antibiotics/antifungal regents to broth or agar?
7. When viewing your plate after incubation; if you notice growth the control quadrant
what would you assume?
8. A colony on an agar plate is a group of cells all from one cell plated during streaking.
True or False? (One colony = one cell)
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Gram Stain
Objective:
1. List and demonstrate the 4 step gram stain procedure
2. Draw and label the bacteria cell wall
Resources: Lecture and lab notes
Supplies:
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Bacteria (gram negative and gram positive)
Water bottle
Slides
Lens paper
Immersion oil
Inoculation loop
Beaker 100ml/500ml
Ethanol
Iodine
Crystal Violet
Red Safrinin
Kimwipe
Microscope
Bunsen burner/striker
Observation:
***View under microscope, draw, record magnification, identify bacteria shape and its growth
pattern or patterns***
1. Look at 4 pre-prepared slides. (Record slide title.)
2. Perform 3 successful gram stains; one gram negative, one gram positive, a mixture.
It is best to start with the gram negative bacteria. This is best because the decolourizing
step can be tricky. If you start with gram negative you can gain an understanding of how
long you need to decolourize for.
NOTE: If you do not get what you expected redo until you get it right.
3. Perform 1 gram stain with unknown bacteria.
Procedure:
Fixing bacteria to a slide:
Broth culture:
-transfer a very small volume of broth containing bacteria to slide. This can be
done with a with a flame sterilized inoculation loop.
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-swipe slide through flame of Bunsen burner.
Bacteria from agar surface:
-place a small drop of water on a slide
-using a sterile inoculation loop scrap bacteria off agar onto inoculation loop.
-mix bacteria with drop of water of slide.
-swipe bacteria through flame of a Bunsen burner.
these steps are the most crucial of the entire gram stain:
-Too much bacteria will make viewing difficult because bacteria will be too close
together.
-Not fixing the bacteria well enough will cause them to be washed away during gram
stain.
-swiping slide through Bunsen burner too much will burn bacteria and kill it.
***swiping slide through Bunsen burner is done to evaporate this liquid. If liquid at
anytime bubbles, your bacteria are dead. This step takes patience***
 put your slide with fixed bacteria balancing across a 400ml beaker with a lab bench
cover underneath.
1. Apply 1-2 drops crystal violet stain for 60secs.
a. Rinse with water to remove excess stain
2. Apply 1-2 drops iodine 30 secs
a. Rinse with water
3. Decolourize with 70% ethanol until run off is clear. Gram positive gram will appear
purple after this step and gram negative will be colourless, therefore don’t
decolourize too long or you may get a false result.
4. Counter stain with Safrinin 1-2 drops for 60secs
a. Rinse with water
b. Blot with Kimwipe (do not wipe!!!)
-using the microscope find a view where you can see bacteria shape and colour. (start
with low power working your way to oil immersion)
5. Place slides in 10% Lysol solution. Inoculation loops are to be flame sterilized at the
end of lab. Throw bench covers in garbage.
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Exercises:
1. Gram negative is what colour? Gram positive is what colour?
2. What is the function of:
a. Crystal violet:
b. Iodine:
c. 70% ethanol:
d. Safrinin:
3. What shape is: Draw each.
a. Cocci:
b. Bacilli:
c. Spirochete
d. Strepto
e. Staphylo
f. Diplo
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Carbohydrate Fermentation (Phenol Red Broth)
Objective:
1. List the biochemical process of fermentation
2. Accurately describe fermentation and its uses in microbiology.
Resources: textbook Supplemented by lecture and lab notes
Supplies:
□ 5 TSA Durham tubes w/ phenol red
□ Bacteria:
 E. Coli
 Alcaligenes faecalis
 Salmonella typhimurium
 Staphylococcus aureus
□ Bunsen burner/striker
□ Inoculation loop
□ Ethanol
□ Beaker
□ Test tube rack
Procedure:
1. Label five tubes with name, date and bacteria name
2. Aseptically inoculate each tube with appropriate bacteria. Leave control tube not
inoculated
3. Place in test tube rack for 18-24 hours in the incubator (48 hrs is used to fit a schedule).
4. Examine tubes before you leave, tomorrow, and the following day. Record observations
in a chart included in your lab report. Multiple observations are important for slow
growers, but due to the alkaline reaction below, readings past 48 hours are suspect, and if
the tube ever becomes yellow, record it as an acid reaction.
5. Examine for evidence of acid (pH) and/or gas production. Some bacteria may only digest
the medium’s proteins producing an alkaline reaction (a pink color mostly at the top), but
this is often difficult to observe unless the tube is left totally undisturbed. Undisturbed
tubes are difficult to obtain in a classroom, so the alkaline reaction is often ignored. Yet,
once the carbohydrate is used up, many bacteria can utilize the protein, shifting the pH of
the medium towards alkaline. This is why readings past 48 hours are suspect.
6. Control tube should be negative
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Exercises:
1. Define Fermentation.
2. Do all organisms produce the same end product from Pyruvate?
3. What is the purpose of phenol red in the culture.
4. What is the function of the Durham tube.
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Catalase Activity
Objective:
1. Desribe and list the uses of the catalase reaction.
2. List the chemical equation for the catalase reaction
3. Accurately demonstrate the catalase test
Resources: textbook Supplemented by lecture and lab notes
Supplies:
□ TSA slants
□ Bacteria:
 Staphylococcus aureus
 Streptococcus faecalis
 Micrococcus luteus
□ Bunsen burner/striker
□ Innocultion loop
□ Pipette/bulb
□ 3% H2O2
Procedure:
***You are responsible for making your own TSA slants for this lab. Refer to lab 2a for
protocol***
Day1:
1. Label TSA slants with name, date and bacteria name
2. Heavily inoculating each bacteria, streak onto TSA slant
3. Incubate 24-48hours
After incubation:
1. Pipette several drops of H2O2 onto TSA slant
2. Look for bubbles:
a. Bubbles = positive test
b. No bubbles = negative test
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Exercises:
1.
2.
3.
4.
What is the importance of catalase in certain bacteria
Do anaerobic bacteria require catalase? Explain.
Write balance equation for degradation of H2O2
What 2 groups of bacteria can be differentiated utilizing a catalase test
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Obtaining a Pure Culture
Objectives:
1. Pick a colony of bacteria from a mixed culture and produce a pure culture of that
bacteria
2. Understand why you need a pure culture to study and why tests do not work if your
culture is unpure.
3. Define: selective media, differential media, enrichment media and when are they
used.
Supplies:
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Mixed culture
2 plates of TSA
plate of each: Columbia CNA agar & EMB agar
Bunsen burner/striker
Ethanol
L rod
Inoculation loop
Gram stain procedure and supplies
Procedure:
1. Using the spreading technique from Lab 2 make a mixed culture on a TSA plate.
2. Incubate for 48hours inverted at 37oC
After 1st incubation:
1. Looking at your plate you should have colonies of bacteria on the plate. Each
colony represents the growth from one bacteria cell. All cells in that colony are the
same strain of bacteria. Using a flame sterilized inoculation loop pick a couple
colonies and streak it on a TSA plate, a Columbia CNA agar, and EMB agar.
2. Incubate for 48hours inverted at 37oC
After 2nd incubation:
1. Look at plates. Record observations in lab report.
2. Perform one gram stain for each plate. Record observations in lab report.
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Exercises:
1. Define: selective media, differential media, enrichment media and when are they
used.
2. TSA plates select for…
3. Columbia CNA agar selects for…
4. EMB agar selects for…
5. What is the purpose streaking a 2nd plate on TSA after the first incubation?
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Acid Fast Stain
Objective:
1. Draw and label an acid fast bacteria
2. Accurately demonstrate an acid fast test
Resources: lecture
Supplies:
□ hot plate
□ Bacteria:
 E.Coli
 Mycobacterium smegmatis or Mycobacterium phlei (5day cultures)
□ 3% acid alcohol (970ml 95% ethanol + 30ml concentrated HCl)
□ carbolfuschin
□ methylene blue
□ waterbottle
□ kimwipe
□ 500ml beaker
□ slides
□ papertowel
□ scissors
Procedure:
1. Prepare smears: heat fix bacteria to a slide. Bacteria cultures will be provided for you in
the front of the class. When taking bacteria from class stocks make sure you are
following proper aseptic technique. (ie. flame sterilized instruments)
2. Cut paper towel to size of slide
3. Place slide on heat block. Place paper towel on top of slide and saturate with
carbolfuchsin.
4. Heat for 5 min
this step requires constant attention. Do not allow paper towel to dry out by adding
additional carbolfuschin but at the same time avoid apply excess stain because run off
with stain the surface of the hot plate.
5. remove and let cool until you can handle slide without burning your fingers.
6. Rinse with water, using a water bottle, for 5 sec over a beaker with a lab bench cover
underneath.
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7. Decolorize with acid-alcohol, add drop by drop until slide remains slightly pink (1030sec)
8. Rinse with water.
9. Counter stain with Methylene blue (30-45sec)
10. Rinse with water (30sec)
11. Blot dry with kimwipe. Do not wipe!!
12. Examine under a microscope with oil immersion. Record observations in lab report.
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Exercises:
1. What was the purpose of heating during the stain?
2. What was the function of the counter stain in the acid fast procedure? What colour was
the counter stain? What was it staining?
3. Are acid fast bacteria Gram positive or Gram Negative? Explain.
4. What makes an organism acid-fast?
5. You should see two colours under the microscope, pink and blue. What does each colour
represent?
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Environmental Affects on Bacterial Growth
Objective:
1. List how temperature, pH, and osmotic pressure affects bacteria individually
and in combination
2. Accurately demonstrate streaking techniques.
Resources: Textbook Supplemented by lecture and lab notes
Supplies:
all tubes/plates will be provided premade in conditions needed
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beaker
ethanol
inoculation loop
Bunsen burner/striker
P1000/blue tips
Temperature:
□ empty, sterile tubes 12/pair
□ 4oC = fridge, 23oC = room temperature, 60oC = water bath, 85oC = water bath,
100oC = boiling H2O
□ broth bacteria:
 Staphylococcus aureus
 Bacillus subtilis
 Pseudomonas aeruginosa
□ Nutrient broth tubes – 13/pair (1/sample +control)
pH:
□ TSA broth tubes at ph 3, 5, 7, 9 (4/pH/pair)
□ Bacteria:
 E.Coli
 Alcaligenes faecalis
 Saccharomyces cerevsisiae
Osmotic Pressure;
□ Nutrient Agar plates
 0%, 0.5%, 5%, 10%, 20%n NaCl
 1 of each/pair
□ Bacteria:
 E.Coli
 Staphylococcus Aureus
 Halobacterium salinarium
Procedure:
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Temperature:
1. Label 5 tubes of each with S. aureus, B. subtilis, P. aeruginase, control
2. Divide one of each into groups: 4oC, 23oC, 60oC, 85oC, 100oC.
3. Pipette 1ml of bacteria into sterile tube. Transfer bacteria using a pipetter. You
will use a P1000 with sterile blue tips. Tips are to be disposed of in a red
biohazard bag to be autoclaved and then thrown away.
4. Place in appropriate condition (fridge, incubator, waterbath) for 15min.
Incubators and waterbaths will be set up prior to lab. Instruction will be given at
the beginning of lab.
5. Allow to come back to room temperature
6. Place in 37oC incubator for 24-48hours. Record tubes with growth.
pH:
1. Label 4 tubes of each condition: TSA broth tubes at pH 3,5,7,9 with E.Coli,
A. faecalis, S. Cerevisiae, control.
2. Inoculate with appropriate bacteria.
3. Incubate in 37oC for 24-48hours. (>48hrs for S. Cerevisiae)
4. Rank tubes in increasing growth of bacteria. Record Observations.
Osmotic Pressure:
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1. divide one plate of each condition: 0%, 0.5%, 5%, 10%, 20% NaCl into
quadrants, label each quadrant: E.Coli, S. aureus, H. salinarium, control.
2. streak with appropriate bacteria
3. Incubate in 37oC for 24-48hours. Record results in lab report.
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Exercises:
1. Explain why some bacteria grow better in some conditions then others. What
evolutionary advantage would this give bacteria?
2. Define:
a. Psychrophiles, mesophiles, thermophiles, acidophiles, neutrophiles,
kalophiles.
3. What is the purpose to the varying concentrations of NaCl in the Osmotic pressure
portion of the lab?
4. pH is a scale of what?
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Antiseptics and Antibiotics
Objectives:
1. To be able to differentiate between antibiotics, antiseptics, and disinfectants and be
able to define each of the above.
2. Evaluate the bacteriostatic activity of antibiotics, antiseptics, and disinfectants.
Supplies:
□ Bacteria
 Staphylococcus epidermidis
 E. Coli
 Pseudomonas aeruginosa
 Mycobacterium smegmatis
□ Antibiotic discs
 Penicillin
 Streptomycin
 Tetracycline
 Chloramphenicol
 Nystatin
 Sulfanilamide
 Erythromycin
□ 70% ethanol
□ 3% Hydrogen peroxide
□ antiseptic mouthwash
□ 0.3% hexachlorophene
□ 6 Mueller-Hinton plates
□ 6 sterile cotton swabs
□ Sterile filter discs
□ Forceps
Procedure:
Antiseptic:
1. With a lab pen divide 2 Mueller-Hinton plates into quadrants. (Write name and date
also)
2. Label each quadrant with the name of the antiseptic you will place in those quadrants.
Do this with both plates.
3. Label one plate S. Epidermis and the other E. Coli
4. Completely streak each plate with the appropriate bacteria
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5. Using sterile forceps dip a sterile filter disc into a antiseptic solution until the disc is
completely moistened.
6. Hold disc above piece of paper towel to allow excess antiseptic solution to drain off.
7. Place disc in the middle of the appropriate quadrant.
8. Repeat steps 5-7 with each antiseptic solution on both plates.
9. Incubate Petri dishes inverted at 37oC for 48 hours
Antibiotics:
1. Divide underside of 4 Mueller-Hinton agar into 6 pie shaped sections.
2. Label each “pie piece” with a antibiotic
3. Label each plate with a bacteria. (S. Epidermis, E. Coli, Pseudomonas aeruginosa,
Mycobacterium smegmatis)
4. Streak each one of your plates with the appropriate bacteria
5. Heat sterilize forceps and transfer one antibiotic disc per pie piece in appropriately
labeled section.
a. FLAME sterilize forceps between each antibiotic disc
6. Invert and incubate plates at 37oC for 48 hours.
After Incubation:
1. Remove plates from incubator and measure in mm the clear zone around each
antiseptic disc and antibiotic.
2. Record results in a table in lab report. Table should include: Bacteria name, antiseptic
or antibiotic, diameter of clear zone.
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Exercises:
1. What is the difference between a disinfectant, an antibiotic and an antiseptic?
2. If there was no “clear zone” around an antibiotic disc what can you say about that
particular strain of bacteria?
3. What is Mueller-Hinton agar?
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The Enterotube
Objective:
1. Write a lab protocol from a commercial protocol
2. Test your protocol by accurately conducting an experiment
Resources: Enterotube protocol
The Enterotube II is a commercial product available from Becton-Dickinson and Company for
identification of enteric rods (Gram-negative bacilli often found in the gut). It has twelve
compartments for twelve tests containing the appropriate media for these tests: glucose, lysine,
ornithine, H2S/indole, adonitol, lactose, arabinose, sorbitol, Voges-Proskauer, Dulcitol/PA, urea
and Simmon’s citrate.
Procedure:
The wire in the Enterotube is used to inoculate the tube. First remove the caps from both ends to
expose the inoculation wire that is sterile. Touch the wire to an isolated colony from a Petri plate
and pull the wire by rotate the other end to inoculate all the compartments with the bacteria.
Make sure the wire is pushed all the way back in to reinoculate the compartments. The wire is
pulled until it reaches the indole compartment where it is broken off (use pliers or another tool).
Following the manufacturer’s directions, flame a needle or a similar tool and perforate the
aerobic compartments and then recap the tube. Incubate at 37 degrees Celsius for 18-24 hours.
Record results as usual for each of the above tests. Reagents are added as needed (e.g. Kovac's
reagent is added to the indole test.)
Lab:
Before lab read the protocol for using the enterotube system.
On lab day come to class and perform experiment.
All supplies needed will be supplied.
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