Semifinal Part C: Bacterial Diagnostics - Topics 15-31

Question 1

What does minimal INHIBITORY concentration (MIC) measure, and how is it determined by dilution method?

Answer 1

Measures effectiveness of bacterioSTATIC antibiotics

Dilution method: prepare various 10-fold dilutions of the ATB in a tube, and attempt to culture bacteria in each of the dilutions. If a sample turns cloudy, it has significant bacterial cultures in it. So, the most diluted non-cloudy sample is the MIC.

However, note that there is still bacteria in it - just not enough to turn it cloudy

Question 2

What does minimal BACTERIOSTATIC concentration (MBC) measure, and how is it determined by dilution method?

Answer 2

Measures effectiveness of bacterioCIDAL antibiotics.

Dilution method: starts off the same as the MIC measurement, but you have to make a culture of your dilutions as well. Usually, the most diluted non-cloudy sample is NOT the MBC, but the first one that’s less diluted than it. You have to go by whichever dilution does not form colonies on culture media.

Question 3

With the disk diffusion method (Kirby-Bauer), you culture a bacterial lawn with discs of antibiotics. How do you determine if the bacteria is susceptible or resistant to the antibiotics?

Answer 3

If the diameter of uncultured area around the disc is > 18mm, the bacteria is susceptible

Diameter between 10 and 18mm means intermediate susceptibility, some resistant

Diameter < 10mm means the bacteria is resistant to the ATB.

Question 4

How does the E test work? How do you determine the MIC from it?
(if you’re not familiar, look up a picture because it’s easier to understand when you visualize it)

Answer 4

You have a strip that has increasing concentrations of an ATB on it. When placed on a culture of bacteria, there should be an ellipsoid (hence “E”) formation around the strip where no bacteria grew.

The MIC is the lowest concentration marked on the test that starts to show no bacterial growth next to it.

Question 5

What is the mode of action for disinfection via alcohol, phenol, and heavy metal ions?

Answer 5

Protein denaturation

Question 6

What is the mechanism for how an autoclave is able to sterilize things?

What are 2 parameters used for autoclave?

Answer 6

Hot saturated steam is more convective than dry air, and it’s often pressurized to be more effective

121 °C, +1 atm for 30 min
134 °C, +2 atm, 10 min

Question 7

What are the parameters used in the Pasteur method for disinfecting liquids?

Answer 7

Wet heat
65 °C 30 min or 85 °C 5 min
Then rapid cooling

Question 8

What are the series of tests to confirm if you have Streptococcae versus other bacteria? And then which species of Strep?

Answer 8

1. Gram stain: should be Gram pos
2. Catalase: Strep are negative (Staph are positive)

From here, can do blood agar (beta hemolytic are pyogenes and agalactiae, alpha are pneumoniae and viridans). Bacitracin: pyogenes is sensitive, agalactiae is resistant. Optochin: pneumoniae is sensitive, viridans is resistant.
Lancefield serology (A = pyogenes, B = agalactiae, D = enterococci)

Question 9

What are the series of tests to confirm if you have Staphylococci versus other bacteria? And then which species of Staph?

Answer 9

1. Gram stain: should be Gram pos
2. Catalase: Staph are positive (strep are negative)
3. Coagulase: aureus is positive, others are negative
4. Novobiocin: saprophyticus is resistant, epidermidis (and others) are sensitive

Question 10

How does the catalase test work?

And the oxidase test?

Answer 10

Catalase: H2O2 is added, catalase breaks it down -> bubbly product. (Note catalase positive bacteria are problems for people with Chronic Granulomatous Disease)

Oxidase: determines if bacteria has cytochrome C oxidase by having a strip with a reagent that turns blue when oxidized by bacteria (most Gram negs)

Question 11

You are testing to see if some unknown enteric bacteria ferments lactose. What steps do you take to determine the bacteria based on lactose fermentation?

Answer 11

Ferments lactose: now perform UREASE test. Positive -> Klebsiela. Negative -> E. coli

Can’t ferment lactose: now check for H2S production. Positive -> Proteus, Salmonella. Negative -> Shigellae

Question 12

How is urease activity tested?

What are some urease positive bacteria?

Answer 12

Urease converts urea into NH3 and CO2. It can be tested by seeing if the pH is raised (because of increasing NH3 concentration) via indicators like phenol red. H. pylori can be tested in vivo with urease that has a radioactive carbon isotope, and it comes out as exhaled CO2.

Urease positive: H. pylori, Proteus vulgaris, Klebsiella

Question 13

What are 2 ways to check for H2S production in bacteria?

Answer 13

TSI: Triple Sugar Iron Medium. Has brilliant sugar (lactose, dextrose, saccharose) + phenol red + iron-salt. Get a black spot where H2S is produced and tests lactose fermentation too.

Hektoen agar: H2S production -> black, similarly tests lactose too.

Most important H2S producing bacteria: Proteus, Salmonella

Question 14

What substrate that produces a color when broken down can be given to check for lactose fermenting bacteria?

What are the most important lactose fermenters?

Answer 14

ONPG: same thing we used in biochem for lac operon. Bacteria with enzymes to process lactose also break this down, creating a colored product.

Most important lactose fermenters: E. coli, Klebsiella, Enterobacter

Question 15

What types of bacteria can EMB help differentiate, and why?

Answer 15

Dyes inhibit growth of Gram pos bacteria (selectivity)

Differentiation: gram negs sorted by ability to process lactose. Lactose fermenters -> dark blue colonies. Non-lactose fermenters -> colorless to pink colonies.

Also contains detergents to prevent swarming.

Question 16

What are 3 major bacterial antigens commonly used for serological testing?

Answer 16

1. O antigen: part of Gram neg cell wall
2. H antigen: flagella (if it exists)
3. K antigen: capsule (if it exists)

Question 17

What occurs with agglutination tests (vs precipitation?)

Answer 17

Agglutination: large particles aggregate as a result of antigen-antibody bonding, generally referring to cells.

Precipitation is composed of antibodies with smaller antigens, often toxins

Question 18

What are some agglutination tests methods? Two examples of specific tests?

Answer 18

Slide, Tube, and Latex (passive) agglutinations used

Two tube agglutination techniques:

1. Gruber-Widal reaction: for Salmonella typhi
2. Weil-Felix Reaction: for Rickettsia prowzekii

Tube and Latex can be used for quantitative measurements (how much Ag is present) by testing various dilutions of serum

Question 19

How do the concentrations of Ab and Ag have to be for a precipitation complex to occur?

Answer 19

[Ag] and [Ab] have to be roughly equal for precipitation. Too much antigen or too much antibody will dissolve the precipitate.

Question 20

In a disc (ringed) precipitation test, where does the precipitation occur?

Answer 20

Antibody is added to a tube, and later antigen is added. A ring/disc of precipitate forms in the middle of the tube at the zone where Ag and Ab are equivalent

(this is a qualitative test)

Question 21

In a disc (ringed) precipitation test, where does the precipitation occur?

Answer 21

Antibody is added to a tube, and later antigen is added. A ring/disc of precipitate forms in the middle of the tube at the zone where Ag and Ab are equivalent.

Question 22

What is the Elek test?

Answer 22

Precipitation test: antitoxin antibodies are on placed on a strip within a culture media for C. diphtheriae. Perpendicular to the strip, 2 lines are made with “control” culture of known diphtheria toxin-producing strains. An “unknown” line is made with the patient’s bacteria to be tested.

Question 23

Why is the complement fixation test relevant in microbiology?

Answer 23

Use it to test for specific antibodies in patient’s serum, one way to see if patient has been infected.

Question 24

How does the complement fixation test work?

Answer 24

1. Take patient’s serum, heat it to destroy own complement
2. Add antigen. If pt has Abs for that Ag, they form a complex
3. Add complement. If there has been a complex, the complement is used up via classical pathway.
4. Add sheep RBCs with anti-sheep RBC Abs. If the complement was used up (pt had antibodies), then the RBCs merely precipitate - no lysis.
   If there was no initial Ag-Ab complex, there is still complement around which lyses the RBCs - turning the solution red.

Question 25

Note: ELISA, immunofluorescent methods, and general principles of serology are topics, but I’m not making cards because they are well-covered in immuno

Answer 25

OK

Question 26

What are some examples of allergic skin tests relevant to microbiology?

Answer 26

Tuberculin: intradermal injection of TB-derived protein. Tests for type IV hypersensitivty response (delayed-type hypersensitivity)

Same thing exists for Brucella, Tularemia, and Leprosy. However, people with the more severe “lepromatous leprosy” do not have reaction to the antigen.

Question 27

What are two awesomely-named susceptibility skin tests used in microbiology?

Answer 27

1. Dick probe: test for erythogenic toxin from S. pyogenes

2. Schick probe: tests for diptheria toxin

Question 28

What are 3 bacteria for which serotypes are important?

Answer 28

1. Chlamydia trachomatis: A-C cause blindness, D-K are the major STI, and L1-L3 cause lymphogranuloma venerum (LGV)
2. Lancefield: Streptococci groups A (pyogenes), B (agalactiae), and D (enterococci)
3. Salmonella enterica have over 2000 serotypes

Question 29

What is ribotyping? How is it performed?

Answer 29

Using bacterial ribosomes to identify strains of bacteria (useful if you want to know transmission origin and epidemiology)

Detect with DNA methods like Southern Blot (like Western blot but for DNA)

Question 30

What is bacteriophage typing? How is it performed?

Answer 30

Like ribotyping, bactiophage typing also helps identify specific strains of bacteria.

It is performed by adding different types of bacteriophages to a culture of bacteria, and observing to see if these particular bacteriophages were able to lyse the bacteria or not (susceptibility test). If the patterns of lysis of different strains (i.e. S. aureus isolated from different patient specimens) are the same, it means nosocomial outbreaks are from one source.

Question 31

What 2 things should always be attempted before initiating or changing antibiotic therapy?

Answer 31

1. Rule out allergies

2. Get a specimen of the bacteria to culture

Question 32

Why would you collect a blood culture versus any other type of culture?

Answer 32

Blood cultures are collected when you suspect bacteremia / septicemia.

Question 33

What are some of the “major rules” for performing a blood culture?

Answer 33

• Of course, be very cautious about contamination. Thoroughly disinfect venipuncture site, wear sterile gloves, etc. Staph epidermidis commonly contaminates blood cultures.
• Take 2 samples of blood: one for anaerobe culture, another for aerobes.
• Withdraw blood from 2 or 3 different sites to exclude skin contamination

Question 34

BONUS: What is the routine way to identify bacterial species that was never taught to us because it makes everything super easy?

Answer 34

MALDI-TOF MS

Matrix-Assisted Laser Desorption/Ionization - Time Of Flight Mass Spectrometry

It’s very accurate, figures out which species it is within a minute, and it’s inexpensive enough that it’s in labs all over the world. Basically everything we learn is outdated because we have this.