Download LAB 2 (Data sheet 3

LAB SAFETY: Open-toed shoes and shorts above the knee are not allowed since a spill
can cause skin irritation. Put equipment back in tote box on the rack in the front of the
class. Before leaving, make sure your area has been disinfected, left clean, and that
the microscope has been put away properly. If it is found not properly put away, you
will receive a note indicating what was wrong with it.
The lab counters must all be wiped down with disinfectant when you come in and
when you leave. You may throw the paper towels into the regular trash afterwards.
Dispose of cover-slips, but the glass slides will be cleaned and re-used. Most fires in
this lab are started from alcohol becoming ignited. Just get away from it, cover it, and
let it burn out. Remember, a lab coat can be taken off and used to put out a fire if
someone’s hair catches fire, etc.
There is no food, drink, gum or water bottles allowed in a micro lab.
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.
For the glassware flasks and tubes, make sure all labels are removed, and all grease
marks are erased first.
If there is a spill, cover it with paper towels, saturate them with disinfectant for ten
minutes, then put those paper towels in the biohazard bucket along with your gloves.
First aid kit is in the cabinet near Andrea’s door.
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INOCULATION TECHNIQUES
To inoculate the sterile tube or plate, there are four different techniques, depending on
what tests you will perform on the subculture.
1) Streak Plate
2) Streak for Isolation
3) Spread Plate
a. A small amount (several drops) of a previously diluted sample is spread
over the surface of a solid medium (Petri dish) using a spreading rod,
which is a sterile glass rod bent at 90°.
4) Pour Plate
a. A small amount of diluted sample is mixed with melted agar and poured
into empty, sterile Petri dishes. A serial dilution of the bacterial sample is
performed first, then a small amount of each dilution is pipetted into the
empty Petri dishes, then the melted agar is added. This allows bacteria to
be inoculate throughout the media.
The spread plate and pour plate techniques are quantitative methods used to determine
the number of bacteria in a sample.
Streak plate method
This is typically used to introduce a 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
used to streak the sample many times in a zig-zag over the surface of a solid culture
medium in a Petri dish. It is streaked from top to bottom.
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Streak for isolation
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. The Streak for isolation technique is used to take the mixture of bacteria and
separate them out into pure cultures to identify each organism.
Use the Petri dish with the four quadrants drawn on the bottom (label each quadrant 1, 2,
3, 4 clockwise). Flame your loop after you inoculate the first quadrant, then drag your
loop ONCE across quadrant 1 and steak it into quadrant 2. Flame your loop and drag it
ONCE across quadrant 2 and streak it into quadrant 3. Flame your loop and repeat for
quadrant 4. Turn your plate counter-clockwise after you perform each streak.
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 a few days, you will see separate colonies in the last
quadrant. If you were not successful, different looking colonies will still be overlapped
in the last quadrant.
What might have gone wrong if your streak for isolation resulted in too many
colonies on the last quadrant? 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. Keeping the plates stored upside down also enables you to
read the important writing on the bottom of the plate.
After you identify 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 colonies that look different, so you end up with one slant for each
colony. You will then have a pure culture of each colony in separate tubes. You would
then let those grow and run some tests on them to determine what organisms are present.
WHEN TO USE AN INOCULATION NEEDLE
If you want to take one pure colony from a plate, use a needle. A loop picks up too much
sample.
Streak for isolation
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EVALUATION OF CULTURES
A. NUTRIENT SLANT CHARACTERISTICS:
1. Is there turbidity (cloudiness)?
2. Is there growth only at the top (pellicle), only at the bottom (sediment), flakes
in the middle (flocculant), or growth throughout (uniform turbidity)?
3. What color is it?
B. PLATE CHARACTERISTICS:
1. MARGINS (look at the surface of the plate)
a. Filiform (smooth edge)
b. Echinulate (serrated edge)
c. Beaded (starts smooth, breaks into individual colonies at top)
2. TEXTURE (look at surface of plate)
a. Flay, dry (Bacillus megatarium)
b. Spreading (proteus, pseudomonas)
c. Crusty, waxy (mycobacterium)
d. Transparent
3. PIGMENTATION (color of plate)
a. Yellow
b. White
c. Green
d. Etc
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
1. orange
2. beige
3. white
CONFIGURATION
round
irregular
rhizoid, arborized
MARGIN
smooth
erose
rhizoid
ELEVATION
convex
flat
raised
umbonate
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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. Intentionally introducing
microbes onto nutrient agar and into nutrient broth is known as INOCULATION, 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.
TYPES OF MEDIA
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, so NA is a good all-purpose
media. It will support the growth of a wide variety of organisms. It is also inexpensive.
There are three types of media used to isolate particular organism
1. Selective media
2. Enrichment media
3. Differential media
Selective media
This type of media selects for a particular type of organism to grow. Selective media
contain chemicals that prevent the growth of unwanted bacteria without inhibiting the
growth of the desired organism. An example is Sabouraud’s Dextrose Agar (SDA), which
has a high sugar content and an acidic pH. SDA is called an ISOLATION MEDIA since
it 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. Toss out the whole jar! Molds and yeast also grow well in an
acidic environment.
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SDA was originally designed to isolate dermatophytes, which are fungi that infect skin,
nails, and hair, and are opportunistic pathogens. 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.
Differential Media
Differential media contains various nutrients that allow the investigator to distinguish one
bacterium from another by how they metabolize or change the media with a waste
product. Some of these media contain a substance that turns yellow if one group of
organisms are present, or red if another group of organisms are present. Therefore, we
can tell which group of bacteria are there by the color the plate turns.
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).
Broths and Slants
Broths are the same nutrients as NA, but no agar. They do not have to be boiled, just
stirred. They are placed in tubes instead of flasks 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).
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MICROSCOPE USE: 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.
PARTS OF A MICROSCOPE: The basic frame of the microscope consists of the base,
stage, arm, and body tube. The substage adjustment knob 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. The ocular is the eyepiece. It magnifies ten times and
focuses the image on the retina. To clean the oculars, squirt alcohol onto lens paper and
wipe, then wipe with dry lens paper.
The left ocular has a diopter adjustment ring that 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
revolving nosepiece holds the objectives. You need to know the following about the four
objectives: The scanning (red ring) objective is the smallest, and magnifies 4x. To find
total magnification, multiply the ocular magnification (they come in 10x and 15x, but we
only have 10x) by the magnification of the objective. What is total magnification on our
microscopes with the scanning objective? Since the ocular is 10x, and the scanning
objective is 4x, 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.
The coarse adjustment knob moves the stage a lot, and the fine adjustment knob moves
it a little. Changing the distance between the stage and the objectives to focus 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. Then rack up the coarse knob 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). To focus from now on, ONLY use the fine adjustment
knob.
PARFOCAL: This term refers to the factory adjustment which means that once you are
focused with the scanning objective, you are focused with all of the objectives (except for
fine adjustment for minor corrections). If you lose focus, always go back to the scanning
objective.
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The CONDENSER takes light from the lamp and makes the rays into a point on the slide. 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. When changing from low to high power, it is
necessary to OPEN the iris diaphragm; increasing magnification requires MORE LIGHT.
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. The
resolving power of a microscope is a function of The numerical aperture (NA) of the lenses and
the wavelength of light
What is the working distance?
Since you start with the lowest power (the shortest objective), as you increase magnification,
working distance decreases because the objectives are taller as you go up in magnification. The
scanning objective has the largest FIELD OF VIEW and the greatest working distance.
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).
BACTERIA SLIDE
Bacteria are small, so they always need 1000x to be seen. The oil immersion lens has the
narrowest depth of field. Why is it called an oil immersion lens? It needs a drop of synthetic
immersion oil.
There are three basic shapes of bacteria:
1. Spiral
2. Cocci (singular is coccus)
3. Bacilli (singular is bacillus; also known as rods)
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.
PUT YOUR MICROSCOPE AWAY LIKE THIS:
 The AC (power) cord is wrapped wrapped loosely behind the scope.
 The arm should face your body, the dust cover in place
 The condenser should be racked up (placed in its highest position). The toggle switch for
power should be off (it is located in the front or side).
 The voltage regulator should be turned to zero or the lowest setting.
 The stage should be racked down
 The scanning objective 4x (red ring lens) should be in place.
 Clean off the oil and fingerprints from the stage and elsewhere
 Wipe the ocular lens with lens paper only.
 Eyepieces should face the blue side of the scope and TIGHTEN the screw a bit
 Clean objectives (not oily on the outside or blurry to look through)
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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.
BACTERIAL CULTURES
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, and you do have to scrub the
patient’s skin while wearing a face mask and sterile gloves
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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.
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ASEPTIC TECHNIQUE FOR TRANSFERRING BACTERIA
General preparations
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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.
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 within arm’s reach so 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 with a blue cone.
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 NONdominant 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, back-and-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
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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. You should always SHAKE a tube of culture broth before removing a
sample because it disperses bacteria that might otherwise have sunk to the bottom of the tube.
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.
To obtain a large inoculum from a broth culture, you need to use a sterile disposable pipettes.
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.
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.
If you are inoculating a tube instead of a Petri dish, pick up the tube with your non-dominate
hand (TUBE hand), take the lid off with the two little fingers of your non-dominant hand (your
LOOP 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.
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.
To inoculate an agar tube, there are several techniques (streak, stab, etc) which will be
described later. When transferring cultures hold the tubes by the base of the tube in your nondominant hand and the sterile loop in your dominant hand.
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.
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STAIN TECHNIQUES
Proper order for staining: Place organism on slide, air dry, heat fix, stain
The purpose of heat fixing 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.
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 acidic dye, also called a negative stain, is used when you want to stain the background
instead of the cells. An example of a negative stain that we will use in lab is Nigrosin.
With negative stains, no heat fixation is necessary; the dye is sticky, so you simply mix
the cells in with the dye and spread it out thinly. Since there is no heat fixation, 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
1) A DIFFERENTIAL STAIN uses several stains; examples are the Gram stain
and the Acid-Fast stain. These are used to find out what category of bacteria are
present.
2) There are also a number of SPECIAL STAINS for viewing spores, capsules, or
flagella.
3) 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.
4) A NEGATIVE STAIN stains the background instead of the bacteria.
DIFFERENTIAL STAINS
A. GRAM STAIN
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.
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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.
B. ACID-FAST STAIN
The results of this stain are recorded as acid-fast or non acid-fast. An example is the
Ziehl-Neelsen 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
If a bacterium is positive for the acid-fast stain, this indicates it has mycolic acid in the
cell wall. Mycolic acid 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 the following:
1. Elderly people or infants
2. AIDS or HIV-infection
3. Immunosuppressing agents for organ transplant recipients
4. Chemotherapy for cancer patients
5. Malnutrition
6. Medicines (some antibiotics)
7. Medical procedures (surgeries, especially implanted joint replacements or internal
fixation hardware such as screws and plates for broken bones)
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SPECIAL STAINS
A. 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
B. 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.
C. FLAGELLA STAIN: Certain bacteria have flagella, which is a whip-like tail
used to help them move. The tail is so thin it is not easily seen with ordinary
stains. A special stain will reveal this structure.
Types of flagellar arrangements
A.
B.
C.
D.
Monotrichous
Lophotrichous
Amphitrichous
Peritrichous
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SIMPLE STAIN
Example: Methylene Blue for cheek cells or bacterial colonies
NEGATIVE STAIN
Example: Nigrosin
If an ion has a negative charge it is called an ANION. A negative stain is one that
contains anions on the chromophore (color portion of the dye). That means the color
portion of the dye has a negative charge (it will also be acidic). 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. The purpose of using a negative stain is to
determine the size and shape (bacilli, cocci, spirals) of an organism and their arrangement
(staphylo, tetrads, strepto, etc). There is no need to heat-fix Nigrosin because the stain
makes the bacteria stick to the slide. Just let it air dry.
There are three types of negative stains:
a) Nigrosin
b) India ink
c) Congo red
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.
Unlike Gram positive bacteria, 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.
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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
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. The iodine forms a complex with crystal violet which reacts with the
peptidoglycan in the cell wall. It keeps the crystal violet from being
washed out by the alcohol
c. Without iodine, all of the stain would wash away.
3. DECOLORIZER
a. Alcohol (ETOH) -This removes the primary stain from some of the cells
(decolorizes some of the cells).
b. This is the most critical step because the alcohol must be left on for just
the right amount of time.
4. COUNTERSTAIN
a. Safranin - This is a red color that stains the cells that became decolorized.
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 negative cells might be purple, Gram + cells might be pink
or purple and pink because some PG has broken down)
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.
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COMPARISONS OF STAINS
G – (PINK)
ACID FAST
Crystal violet
Crystal violet
Iodine
ETOH or
acetone:
Cell is purple
Safranin:
Cell is purple
Iodine
ETOH or
acetone:
Cell is clear
Safranin:
Cell is pink
Carbol
Fuschia
Heat
Acid alcohol
G+ (PURPLE)
PRIMARY
STAIN
MORDANT
DECOLORIZER
COUNTERSTAIN
Methylene
Blue
CAPSULE
STAIN
Malachite green
Heat
Water
Safranin:
Spores are
green
Cell is pink
SPECIAL MEDIA for Isolating Bacteria
MANNITOL SALT AGAR (MSA)
The MSA plate contains a lot of salt. Only Gram positive organisms can tolerate this.
EOSIN AND METHYLENE BLUE (EMB) PLATE
Contains those two dyes that inhibit Gram positive bacteria, but are attractive to Gram
negative bacteria.
MSA and EMB agar are therefore SELECTIVE MEDIA because only certain types of
bacteria will grow on them. All of your organisms will grow on the Nutrient agar, so it is
not selective.
MSA also contains a sugar called mannitol. Only certain organisms will use the sugar. If
the sugar is used, the waste product of its metabolism is an acid. The red color in the plate
is from methyl red, a pH indicator that will turn yellow if acids are present. If the media
turns yellow next week, the organism fermented the mannitol sugar.
We can grow more than one organism on that plate and see that one ferments mannitol
and the other does not. That means that MSA is also DIFFERENTIAL MEDIA.
Therefore, MSA is selective AND differential media, while EMB is just a selective
media. Salt makes MSA selective, and mannitol makes it differential.
BLOOD AGAR
This is classified as ENRICHED MEDIA. In this media, sheep’s blood is added to
nutrient agar. This plate would grow organisms that are fastidious (picky eaters), such as
pathogenic bacteria like Streptococcus pyogenes (causes strep throat). We could also see
whether the organism demonstrates alpha hemolysis (blood turns green), beta hemolysis
(blood turns clear), or gamma hemolysis (no change in blood color).
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ENUMERATION OF BACTERIA
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 (aka estimating cell density), 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. 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.
Samples are placed in special tubes called cuvettes, which are test tubes of optically
pure glass that will not absorb light like regular glass test tubes. These are placed into
the spectrophotometer, and the transmission is read for 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%. A transmission reading should be
between 1-99%. The next sample is placed in the machine (it does not need to be
blanked and zeroed anymore), the reading is obtained, and likewise for all the
samples.
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Once you have your transmission readings, turbidity is recorded as a sample’s
optical density.
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 to OD). 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.
2. STANDARD PLATE COUNT (SPC)
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.
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
Problem:
We have to use the plate in the 30-300 range.
If the plate we count has 200 colonies, and it was taken from the plate that was diluted to
1: 100,000, what is the number of living organisms in the original sample?
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
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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
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.
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. Which organism
had the highest OD for pH 7? That organism grew the best in that pH, so it was
a neutrophile.
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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
chemicals, heat, and UV light exposure for a longer time. You could design an
experiment to expose plates of two organisms to UV light for various periods to see
how long it takes to kill each type of bacteria with UV light. Gram positive rods are
the only bacteria that make endospores. An example is Bacillus.
Staph aureus (Gram negative rod) is exposed to a various number of seconds of UV
light
Bacillis (Gram positive rod) 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.
FORMULA TO MEASURE RESISTANCE TO UV LIGHT
Time to kill Bacillus ÷ time needed to kill Staph.
Note: to apply this formula to 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.
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). Nonpathogenic 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. Overall, bacteria are more heat resistant that most
other forms of life, but they can only tolerate a certain amount of heat. Generally, if
heat is applied, microbes are killed; if cold temperatures are used, microbial growth
is inhibited.
Psychrophiles: like freezing cold temperatures
Mesophiles: prefer anything from room temperature to body temperature (25-40 °C).
Thermophiles like it quite hot (45-65 °C)
Hyperthermophiles: like extreme heat, such as found in volcanoes.
Most human pathogenic bacteria are mesophiles.
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EVALUATION OF ANTISEPTICS AND DISINFECTANTS
An antiseptic is used on tissue, not inanimate objects. There are two kinds of
antiseptics: topical (like Bactine) and oral (like Scope and Listerine).
Disinfectants are chemicals that are used on inanimate objects only.
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.
HAND SCRUBBING EXPERIMENT
A BACTERIOSTATIC agent is one that causes temporary inhibition of growth of bacteria.
A BACTERIOCIDAL agent is one that kills bacteria.
An ANTISEPTIC is a chemicals used on living tissue to decrease the number of
microbes
A DISINFECTANT is a chemical used on NON-living tissue to decrease the
number of microbes
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. Washing with soap and water, even for a short
time, will remove some transient organisms.. Resident organisms are those
which stay on us, even after we wash with soap and water. 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 transient organisms. Washing with soap and water removes many
more transient organisms and some resident organisms. Resident organisms cannot
be completely removed from the skin; it is impossible to sterilize living tissues.
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.
22
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.
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