1. Lab 4
Microlab 2421

2. quiz
• what structure is the gram stain targeted or
stain?
• Why do you not heat fix on the capsule stain.

3. quiz
• what structure is the gram stain targeted or
stain?
peptidoglycan Cell wall
• Why do you not heat fix on the capsule stain.
Because it might shrink the cell where it looks
like a non capsulated cell, look capsulated, Or
it destroys the capsule.

4. Bacillus anthracis
• This is sporulating microorganism that it occurs most
commonly in soil.
• This organism has been use countless in bioterrorist attacks
including Amerithrax where Bacillus anthracis was sent in
the mail to a senator, and several reporters .
• This organism usually attacks a individual thought the
breathing in spores where it will cross the lungs epithelium
in to the organism blood where it then will be intake by a
macrophage, where it will then become a vegetative cell
where it starts to replicate inside the macrophage.

5. Answers
• Questions
• 1. How many colonies did you count on your “spread plate”?
Calculate how many bacteria were in the mix prior to your dilution?
(Hint: remember the volume that you added to the plate and the
dilution you carried out. Give your answer in the format of colony
forming units/ml (CFU/ml). Count the colonies from all of your
dilutions if possible. Your calculations should give you the same
answer or very close. The equation that is used
• Colonies =X
X/10^-8=_Y____ cfu/(1/10)ml
Y/.10=cfu/ml
• 2. Why did you test 3 different dilutions of culture? Do not write
that the instructions told you to do so.
• This was to see how the different dilutions will have different
isolated colonies

6. • 3. Did you have isolated colonies of all three types on your streak for isolation? Did
you have isolated colonies of all three types on your spread plate? Which method
of isolation worked better streaking for isolation or spread plate? Do you know
why?
• 4. Why is a colony considered “pure”?
• Because all the organism in the colony are clones of each other, because they are
all derive from each cell
• 5. Why did you heat the inoculating loop in between streaking for isolation?
• To kill the bacteria so each time you streaked it would then dilute the bacteria
• 6. What is the difference between a “contaminated” culture and a “mixed”
culture? What was the mixture that you were given?
• A contaminated culture the organism is not known in a bacteria of 2 or more.
Mixed is where 2 or more bacteria are known.
• 7. Why did you use sterile saline to dilute your bacteria culture? (Hint: what would
have happened if you used pure water?)
This was because if you had pure water then it would lyses the cell thought osmotic
pressure increasing and breaking open the cell.

7. Today
• Three different stains that will be used today.
• Gram stain
• Capsule stain
• Spore stain

8. Gram stain
• This is a differential stain
• What is a differential stain ?
• This is a stain that differentials some structure
of a bacteria.
• Was invented by Hans Christian Gram.
• Cristal violet is the primary stain
• Grams iodine is the mordant
• safranin secondary stain.

9. Gram stains
• Why is the Gram stain important?
• Because the gram stain tells doctors how to
treat the infection. It also tells microbiologist
how to work with the bacteria.

10. Gram +/-

11. Gram stain
• Procedure for Gram Staining
• The Gram stain is not difficult to carry out, however, you must use care when performing this
technique. If you decolorize too long, or you do not rinse the 95% alcohol off soon enough, even
the Gram + will decolorize. Please pay attention and do not rush this exercise. As a control, one
slide will have a mixture of Gram + and Gram – bacteria. If the stain is performed properly, the mix
should contain both purple and pink bacteria. Do not leave the laboratory today without
accomplishing this task.
• 1. Label your slide as above and add H20 for 2 samples per slide.
• 2. Using sterile technique, add a small amount of bacteria to each side. Each slide will have a single
bacterium on one side and a mix of 2 bacteria (one Gram + and either Klebsiella pneumonia or the
Pseudomonas aeruginosa) on the other. Allow the slide to air dry and heat fix as above.
• 3. Cover the slide with crystal violet and allow it to stain for 2 minutes.
• 4. Rinse the stain off with H20.
• 5. Add Gram’s iodine to the slide for 1 minute.
• 6. Rinse the iodine off with H20.
• 7. Hold the slide at an angle over the sink or staining rack and “decolorize” with 95% alcohol for 5
sec. Quickly rinse with H20.
• 8. Add the safranin for 3 minutes and rinse with H20. Report your observations below.

12. Gram stain

13. Endospore
• This stain uses malachite green to cook in to the
cell. This is the primary stain
• Safranin was the counter stain
• The spore can be referred to as
• terminal(at one end)
• subterminal( near but not at the end of the cell)
• Central
• Spores can also be referred to as free spores if its
not in the vegetative cell.

14. Endospore stain
• Procedure for endospore staining:
• 1. Slides have already been prepared by the aseptic transfer of a sample of Bacillus
brevis and a sample of Pseudomonas aeruginosa to a slide. The samples were air
dried but you NEED to HEAT fix the specimen.
• 2. Tear a paper towel just smaller than the size of the slide and cover the heat-
fixed slide with the toweling. Flood the slide with malachite green, a basic stain
which will stain the vegetative cells as well as the endospores.
• 3. CAREFULLY, using the clothes pins, hold your slide over the Bunsen burner just
out of reach of the flame. Alternatively, have a beaker of boiling water and use this
as a heat source. Do not make the dye boil. If you notice that the malachite green
is drying up, add more. Heat the slide for 5 minutes.
• 4. Cool the slide before DESTAINING gently with H20 for 30 sec.This will decolorize
the vegetative cells.
• 5. Counterstain the cells with safranin for 2 minutes. The counterstain is also called
the secondary stain.
• 6. Rinse the slide with H20, blot dry and observe with the oil immersion objective.
Draw your observations. Where are the endospores; subterminal, terminal, etc. Do
you see free endsopores?

15. Endospore stain

16. Capsule Stain
• There are two different stains the Maneval and the
Anthony stain.
• What one are we using today
Manevel
This stain uses Congo red as a ph indicator s when you
add Maneval solution to the Congo red it changes
Congo red to a purple or blue.
• What are two reasons why the capsule is beneficial to a
bacteria?
• It allows for adhesion, and doesn’t allow for a
macrophage to phagocytosis.

17. Capsule stain
• Procedure for CAPSULE stain
• 1. Place a few drops of Congo red on a clean slide. Do NOT use a
water drop in this sample preparation. 2. Mix in a small amount of
Klebsiella pneumonia. 3. AIR DRY. DO NOT HEAT FIX. (Heating the
slide will destroy the polysaccharide capsule and cause non-
encapsulated cells to shrink and appear encapsulated) 4. Place the
slide on a rack of the staining tray. Gently cover the smear with the
Maneval solution and wait 5 minutes. 5. Lift the slide and gently
pour off excess stain and GENTLY rinse with water. If you hit the
slide with a strong force of water, you may remove the cells and
stain. 6. Place slide on Kimwipes to soak up the excess water. Do
not blot, the slide needs to air dry.
• 7. Examine the slide with an oil immersion lens at 1,000X
magnification. Repeat with E. coli.

18. Capsule stain

19. Next week
• Study for next weeks quiz