Download VCLab 5 Spore stain and acid fast stain

VCLab 5 Spore stain and acid fast stain
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
2. DORNER METHOD
For this method, we will have to make a spore suspension:
a. Place 5 drops of sterile water in a test tube (one tube per table).
b. Add several loopfuls of bacillus to the tube of water.
c. Add 5 drops of carbolfuscian.
d. Heat in a beaker of boiling water for 10 minutes.
e. Cool.
f. Mix several loopfuls of bacteria from the suspension in one drop of nigrosine on the
slide.
g. Either spread the nigrosine with another slide or smear the drop into a large circle
with your loop.
h. After you sterilize your loop for the last time, wipe it off with a paper towel to
remove the nigrosine.
i. Air dry, do not heat fix.
j. Examine under oil
The cells will look clear with a red endospore inside.
You may also see free endospores stained red.
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?