Semifinal Part C: Bacterial Diagnostics - Topics 1-15

Question 1

Steps for preparation and fixation for bacterial smears

I simplified them as 4 steps but you can list them other ways

Answer 1

1. Use a clean slide and burn off anything on it (5 times over Bunsen burner flame)
2. Also flame the loop probe thing until it is hot along its entire length, then allow it to cool
3. Liquid sample: use loop to smear small droplet in circle. Solid sample: put a water droplet on, then add sample with loop
4. Let it dry, and then “fix” the sample by burning the bottom of the slide 3 times quickly

Question 2

What are the 3 native preparations we learned?

Answer 2

1. Wet mount preparation
2. Vital staining
3. Hanging-drop preparation

Question 3

What are the 3 steps for a Wet Mount preparation?

Answer 3

1. Place a small drop of suspension (with bacteria, protozoa) in center of slide OR disperse a small quantity of specimen on a saline drop
2. Cover with the coverslip
3. Examine with the microscope under low power

Question 4

What are 3 steps for Vital Staining?

Answer 4

1. Make a wet mount preparation
2. Add methylene blue (which stains the cells without killing them) to the edge of the coverslip
3. Wait 10 minutes, then examine under low power
   Living microorganisms are blue

Question 5

What are 5 steps for Hanging Drop preparation?

mine is different than the Semmelweis instructions because their English is terrible and this way makes more sense

Answer 5

1. Place a drop of bacteria/protozoal suspension on the cover slip
2. Spread vaseline or wax around the rim of the slide’s cavity so that it sticks
3. Pick up the slide, flip it upside down, and then push it down on the cover slip to make it stick
4. Flip the slide and now the drop will be “hanging” from the coverslip
5. Examine under low power: motile microbes dart through the microscope field

Question 6

What are the steps for Gram staining?

I simplified them to 5 steps

Answer 6

1. Stain a fixed specimen with crystal violet for 2 min, then rinse with water
2. Stain with Iodine (Lugol) for 1 minute, then rinse
3. Rinse with 96% alcohol until alcohol coming off is colorless, then rinse with water again
4. Counterstain with Fuchsin or Safranin for 1 min
5. Dry with filter paper

Question 7

What type of microscopy should be used for Gram staining?

How do Gram positives vs negatives appear, and why?

Answer 7

Should use oil immersion light microscopy

Gram positives are blue-purple because alcohol does not wash out the crystal violet from their thick peptidoglycan layer

Gram negs: the crystal violet washed out, so they are stained red by the fuchsin/safranin counterstain

Question 8

Which bacteria are Gram stain NOT useful for?

Answer 8

Spirochetes are too slim for Gram stain, but would be Gram neg - have LPS

Intracellular pathogens with abnormal cell walls: Mycoplasma, Chlamydia, and Rickettsia

Question 9

What is the purpose of Neisser stain?

What are the steps?

Answer 9

Purpose: Show metachromatic or volutin granules in Corynebacteria diptheriae
Steps:
1. Stain fixed smear from Cornyebacteria culture with 2:1 mixture of Neisser I and Neisser II dyes (contain Crystal violet and Methylene blue) for 10 minutes, then rinse
2. Stain with crysoiodide for 2 min
3. Dry with blotting paper
-> Club-shaped rods (“maracas”) with metachromatically purple volutin granules

Question 10

When would you use Acid-Fast staining? Why?

Which reagents are used, and what is the color of acid-fast stained bacteria?

Answer 10

Acid-fast (Ziehl Neelsen) stains lipid substances in the cell walls (mycolic acids) of Mycobacterium and weakly in Nocardia.

Reagents: carbol fuchsin, acid alchool, and methylene blue. Stain bright red (fuchsia)

Question 11

What are the steps for Acid-Fast staining?

5 listed

Answer 11

1. Flood slide with carbol fuchsin
2. Steam or apply Bunsen burner heat under slide 3x
3. Add acid and alcohol, then wash with water
4. Counterstain with Methylene blue, then wash with water
5. Place filter paper to dry
   Mycobacterium take up the carbol fuchsin, especially with heat. Alcohol and acid won’t wash it out, and so mycobacterium stays bright red while other bacteria are counterstained by methylene blue

Question 12

How do you perform capsule staining?

4 steps listed

Answer 12

1. Smear culture and cover with India ink (or nigrosin)
2. Allow it to dry then heat fix
3. Stain with Fuchsin for 1 min, then rinse with water
4. Allow to dry then observe with oil immersion
   Capsule is “negatively stained” - India ink makes dark background against which the capsule can be seen like an “unstained halo” surrounding the cell

Question 13

What stain is used to identify spores? How does it work?

Answer 13

Schaeffer-Fulton stain

1. Bacteria put on slide, fixed with heat.
2. Slide is then steamed by being placed over a water bath
3. While being steamed, Malachite green is placed on the slide, which penetrates through the endospores and stains them green. Stain for 5 min.
4. Remove the slide from the steam, cool down, and rinse with water
5. Counterstain with safranin to turn other microorganisms red

Question 14

What kind of bacteria is Dark Field Microscopy used for? How does it work?

Answer 14

Spirochetes (Trepenoma, Borrelia, Leptospira) - too small for Gram staining

Uses a special condenser that makes a dark field that contrasts against the highlighted edge of specimens

Question 15

What are 4 PHYSICAL methods of sterilization?

Answer 15

1. Dry heat: flame, oven etc.
2. Wet heat: pasteurization, autoclave, boiling
3. Filtration: filters may remove bacteria but not viruses
4. Irradiation: ionizing, UV, sometimes infrared

Question 16

How is sterility assessed?

Answer 16

No growth on any medium, which confirms the total absence of viable microorganisms (including spores)

Question 17

What are 3 requirements for growth to be considered when preparing culture media?

Answer 17

1. Chemical composition: nutrition with E sources, carbon, nitrogen, etc. Water and pH too.
2. Temperature: most bacteria grow between room temperature and body temperature, but there are exceptions
3. Aeration: important ones are mostly facultative anaerobes, but must consider anaerobes, obligate aeorbes, and microaerophilic

Question 18

What are two bacteria species that we need to know that don’t grow on culture media?

Answer 18

1. Trepenoma pallidum (syphillis) - labs “culture” it in rabbit testis
2. Rickettsia: cell cultures

Question 19

What are the 3 main agar media?

Answer 19

1. Agar: meat bouillon + agar (dried algae that forms gel)
2. Blood Agar: regular agar + 5% sheep blood
3. Chocolate Agar: take blood agar and boil it, turns chocolate-colored. Better for more fastidious bacteria that need their food broken down for them

Question 20

What are selective and differential media? What’s the most important one to know that is both selective and differential?

Answer 20

Selective: Allows growth of only select strains, inhibiing others
Differential: Contains a special color indicator that helps identify the chosen strain

Eosin-Methylene Blue (EMB) - inhibits Gram positive (selective), and the Gram negatives are differentiated by how they ferment lactose. Lactose fermenters (like E. coli) turn the culture green to dark purple. Bacteria that don’t ferment lactose (like Shigella) turn it pink. EMB commonly used for fecal samples.

Question 21

What is one example of a transport media? (NOT culture transport media)

Answer 21

Transport media: Stuart Transport Media - used if specimen cannot be cultured immediately. Preserves microorganism balance, prevents overgrowth.

It’s a non-nutrient soft agar gel containing reducing agent to prevent oxidation, and charcoal to neutralize bacterial inhibitors.

(Transport media must be able to keep the organism for 48 hours)

Question 22

What is one example of a transport culture media?

Answer 22

Uricult: used if the microorganisms can be cultured immediately.

Question 23

How do you prepare a “surface streak culture” versus a “bacterial lawn?”

Answer 23

Surface streak: swab all over 1/2 of petri dish, then do 3 streaks over 1/4 of the dish, and do a zig-zag over the remaining 1/4 (you may see variation in this method)

Bacterial lawn: keep swabbing half of the petri dish then rotating it 90 degrees, until the entire petr dish has been evenly swabbed.

Question 24

Definition of pure culture:

Answer 24

Pure culture: population of cells growing in the absence of other species types, and the cells are genetic clones of one another

Question 25

Definition of “strain” and “isolate”

these are both nouns in this biology / microbiology sense

Answer 25

Strain: genetic variant or subtype of microorganism, i.e. one particular strain of the influenza virus

Isolate: a pure culture derived from a heterogenous, wild population of microorganisms

Question 26

What is “germ count” versus “colony-forming unit (CFU)?”

Answer 26

Germ count: the number of ALL bacterial/fungal cells in the culture, water, food, etc.

CFU: number of LIVING microorganisms. Usually measured CFU/mL

Question 27

How do you measure CFUs?

Answer 27

1. Take your sample and make a series of dilutions that are 10x more and more diluted (10^-1, 10^-2, and so on)
2. Make cultures for all of your dilutions and compare them. The more diluted samples should have progressively fewer colonies.
3. Calculate the number of colonies per dilution (i.e. 24 colonies and 10^-6 dilution = 24,000,000 microorganisms in the sample). Should average them out

Question 28

What is a PHYSICAL method to remove enough oxygen for obligate anaerobic bacteria to grow?

Answer 28

Anaerostat: pump air out of the metal container containing culture, and pump in a mixture of other gases

Question 29

What is a CHEMICAL method to remove enough oxygen for obligate anaerobic bacteria to grow?

Answer 29

Gas-Pack Jar: catalytic reagents extract oxygen from the air in a closed space, also generating CO2 rich air. May also just reduce the O2 concentration for microaerophilic bacteria

Question 30

What is a BIOLOGICAL method to remove enough oxygen for obligate anaerobic bacteria to grow?

Answer 30

Inoculate obligate aerobic bacteria with the obligate anaerobes, but seal it off so that the obligate aerobes consume all the O2
(Fortner technique)

Question 31

What are two microaerophilic bacteria, and what is a culture used for them?

Answer 31

Campylobacter jejuni and Helicobacter pylori. They have to have O2 about 5% and CO2 about 5%.

Use Skirrow culture media: blood agar with yeast, peptones, dextrose, and VPN antibiotics to suppress growth of other bacteria

Question 32

What are 3 major obligate intracellular bacteria?

How can they be cultured?

Answer 32

Mycoplasma, Chlamydia, Rickettsia

Can culture them with cell cultures (like HeLa), with chicken embryo yolk sacs, or in living animals

TB is cultured via Löwenstein-Jensen (solid) medium: malachit-green, egg, potato-flour, glycerol, asparagine, inorganic salts

Question 33

What are 3 examples of laboratory animals used in medical microbiology?

Answer 33

1. Armadillos used to study leprosy because they are commonly carriers of it (especially the 9-banded armadillo from Texas). Leprosy requires low body temp.
2. Romer probe: a culture of C. diphtheriae is mixed in water and injected into each of two guinea pigs, one of which has received antitoxin previously
3. T. pallidum is cultivated in rabbit testis

Question 34

What is the Quelling reaction?

this should have been earlier in the deck

Answer 34

It’s a test for capsule by adding anti-capsular antibodies, which shows up under light microscopy by an increase in refractivity around encapsulated bacteria.

Reminder of some encapsulated bacteria: S. pneuomoniae, Klebsiella pneumoniae, N. meningidits, Salmonella, the encapsulated E. coli (K capsule) etc