Semifinal Part C: Bacterial Diagnostics - Topics 1-15 Flashcards
Steps for preparation and fixation for bacterial smears
I simplified them as 4 steps but you can list them other ways
- Use a clean slide and burn off anything on it (5 times over Bunsen burner flame)
- Also flame the loop probe thing until it is hot along its entire length, then allow it to cool
- Liquid sample: use loop to smear small droplet in circle. Solid sample: put a water droplet on, then add sample with loop
- Let it dry, and then “fix” the sample by burning the bottom of the slide 3 times quickly
What are the 3 native preparations we learned?
- Wet mount preparation
- Vital staining
- Hanging-drop preparation
What are the 3 steps for a Wet Mount preparation?
- Place a small drop of suspension (with bacteria, protozoa) in center of slide OR disperse a small quantity of specimen on a saline drop
- Cover with the coverslip
- Examine with the microscope under low power
What are 3 steps for Vital Staining?
- Make a wet mount preparation
- Add methylene blue (which stains the cells without killing them) to the edge of the coverslip
- Wait 10 minutes, then examine under low power
Living microorganisms are blue
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
- Place a drop of bacteria/protozoal suspension on the cover slip
- Spread vaseline or wax around the rim of the slide’s cavity so that it sticks
- Pick up the slide, flip it upside down, and then push it down on the cover slip to make it stick
- Flip the slide and now the drop will be “hanging” from the coverslip
- Examine under low power: motile microbes dart through the microscope field
What are the steps for Gram staining?
I simplified them to 5 steps
- Stain a fixed specimen with crystal violet for 2 min, then rinse with water
- Stain with Iodine (Lugol) for 1 minute, then rinse
- Rinse with 96% alcohol until alcohol coming off is colorless, then rinse with water again
- Counterstain with Fuchsin or Safranin for 1 min
- Dry with filter paper
What type of microscopy should be used for Gram staining?
How do Gram positives vs negatives appear, and why?
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
Which bacteria are Gram stain NOT useful for?
Spirochetes are too slim for Gram stain, but would be Gram neg - have LPS
Intracellular pathogens with abnormal cell walls: Mycoplasma, Chlamydia, and Rickettsia
What is the purpose of Neisser stain?
What are the steps?
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
When would you use Acid-Fast staining? Why?
Which reagents are used, and what is the color of acid-fast stained bacteria?
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)
What are the steps for Acid-Fast staining?
5 listed
- Flood slide with carbol fuchsin
- Steam or apply Bunsen burner heat under slide 3x
- Add acid and alcohol, then wash with water
- Counterstain with Methylene blue, then wash with water
- 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
How do you perform capsule staining?
4 steps listed
- Smear culture and cover with India ink (or nigrosin)
- Allow it to dry then heat fix
- Stain with Fuchsin for 1 min, then rinse with water
- 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
What stain is used to identify spores? How does it work?
Schaeffer-Fulton stain
- Bacteria put on slide, fixed with heat.
- Slide is then steamed by being placed over a water bath
- While being steamed, Malachite green is placed on the slide, which penetrates through the endospores and stains them green. Stain for 5 min.
- Remove the slide from the steam, cool down, and rinse with water
- Counterstain with safranin to turn other microorganisms red
What kind of bacteria is Dark Field Microscopy used for? How does it work?
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
What are 4 PHYSICAL methods of sterilization?
- Dry heat: flame, oven etc.
- Wet heat: pasteurization, autoclave, boiling
- Filtration: filters may remove bacteria but not viruses
- Irradiation: ionizing, UV, sometimes infrared
How is sterility assessed?
No growth on any medium, which confirms the total absence of viable microorganisms (including spores)
What are 3 requirements for growth to be considered when preparing culture media?
- Chemical composition: nutrition with E sources, carbon, nitrogen, etc. Water and pH too.
- Temperature: most bacteria grow between room temperature and body temperature, but there are exceptions
- Aeration: important ones are mostly facultative anaerobes, but must consider anaerobes, obligate aeorbes, and microaerophilic
What are two bacteria species that we need to know that don’t grow on culture media?
- Trepenoma pallidum (syphillis) - labs “culture” it in rabbit testis
- Rickettsia: cell cultures
What are the 3 main agar media?
- Agar: meat bouillon + agar (dried algae that forms gel)
- Blood Agar: regular agar + 5% sheep blood
- Chocolate Agar: take blood agar and boil it, turns chocolate-colored. Better for more fastidious bacteria that need their food broken down for them
What are selective and differential media? What’s the most important one to know that is both selective and differential?
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.
What is one example of a transport media? (NOT culture transport media)
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)
What is one example of a transport culture media?
Uricult: used if the microorganisms can be cultured immediately.
How do you prepare a “surface streak culture” versus a “bacterial lawn?”
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.
Definition of pure culture:
Pure culture: population of cells growing in the absence of other species types, and the cells are genetic clones of one another
Definition of “strain” and “isolate”
these are both nouns in this biology / microbiology sense
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
What is “germ count” versus “colony-forming unit (CFU)?”
Germ count: the number of ALL bacterial/fungal cells in the culture, water, food, etc.
CFU: number of LIVING microorganisms. Usually measured CFU/mL
How do you measure CFUs?
- Take your sample and make a series of dilutions that are 10x more and more diluted (10^-1, 10^-2, and so on)
- Make cultures for all of your dilutions and compare them. The more diluted samples should have progressively fewer colonies.
- 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
What is a PHYSICAL method to remove enough oxygen for obligate anaerobic bacteria to grow?
Anaerostat: pump air out of the metal container containing culture, and pump in a mixture of other gases
What is a CHEMICAL method to remove enough oxygen for obligate anaerobic bacteria to grow?
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
What is a BIOLOGICAL method to remove enough oxygen for obligate anaerobic bacteria to grow?
Inoculate obligate aerobic bacteria with the obligate anaerobes, but seal it off so that the obligate aerobes consume all the O2
(Fortner technique)
What are two microaerophilic bacteria, and what is a culture used for them?
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
What are 3 major obligate intracellular bacteria?
How can they be cultured?
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
What are 3 examples of laboratory animals used in medical microbiology?
- Armadillos used to study leprosy because they are commonly carriers of it (especially the 9-banded armadillo from Texas). Leprosy requires low body temp.
- 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
- T. pallidum is cultivated in rabbit testis
What is the Quelling reaction?
this should have been earlier in the deck
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
