Exam 2

Question 1

Explain the standard plate count and its purpose

Answer 1

It tells how many colony forming units were in the original sample. To back calculate how many colonies were in a dense original sample.

Question 2

What does TNTC stand for?

Answer 2

Too numerous to count

Question 3

The substance on which an enzyme acts. Medium where organism grows. What make the medium differential. Relies on their differing abilities to perform specific chemical reactions and do them in a way that can be observed.

Answer 3

Substrate

Question 4

Exotoxins that are able to destroy red blood cells and hemoglobin.

Answer 4

Hemolysis

Question 5

Enzyme that operates in the external environment after being secreted from the cells

Answer 5

Exoenzyme

Question 6

An antimicrobial substance produced by a microorganism such as a bacterium or fungus

Answer 6

Antibiotic

Question 7

What is a biofilm? Why is it a concern in hospitals?

Answer 7

A layer of bacterial cells adhering to and reproducing on a surface. Used commercially for acetic acid production and water purification. Ex. dental plaque is a type of biofilm. Some aren’t easy to get rid of and can be antibiotic resistant. This means some meds won’t work for people who are sick from biofilm infections. Especially concerning for patients with implanted medical devices like pacemakers, catheters, prosthetic joints, mechanical heart valves, endotracheal tubes, even contact lenses. Biofilms that form on these devices can potentially release toxins and can be highly resistant to antibiotics especially if formed in a patient’s body

Question 8

A staining technique that is used to determine morphology and cellular arrangement in bacteria that are too dictate to withstand heat-fixing. Produces minimal cell shrinkage. Dye solution used is chromogen acidic w/ neg charge. Put drop of acidic stain at one end of clean slide. Add organism and emulsify with a loop. Place second clean slide on surface of first slide and draw it back into the drop then push back to other end. Air dry, view in microscope. NO HEAT FIXING! Under microscope like b. megaterium cells are stained against dark background. Circular objects are bubbles. The b. megaterium look like white sausage links.

Answer 8

Negative stain

Question 9

Stain that uses crystal violet, iodine, decolorizer, counterstain (safranin). Gram positive=purple, gram negative = pink. Begin with heat fixed emulsion or air dried. Cover smear with crystal violet 1 min, rinse w/ distilled water, gram’s iodine, rinse, ethanol/decolorizer until runoff is clear; counterstain with safranin. Blot dry w/ bibulous paper. Observe under oil immersion.

Answer 9

Gram stain

Question 10

Stain used to detect cells capable of retaining a primary stain when treated with an acid alcohol. M. tuberculosis. Presence of mycolic acids (waxy, gives these cells higher affinity for primary stain and resistance to decolonization) in cell walls of organisms. Cover heat fixed smear with bibulous paper. Add ZN carbolfuchsin (purple) stain and steam for 5 minutes. Remove paper. Decolorize with acid-alcohol until runoff is clear. Counterstain with methylene blue. Rinse distilled water. Blot dry with bibulous paper.

Answer 10

Acid fast stains

Question 11

Stains around the cells because capsules are made of mucoid polysaccharides or polypeptides that repel most stains. Typically an acidic stain like Congo red stains background and basic stain that colorized the cell proper are used. The capsule remains un stained and appears as a white halo between the cells and the colored background. Used to detect cells capable of producing an extracellular capsule. Anthrax. Capsule production increases virulence in some microbes by making less vulnerable to phagocytosis. No heat fixing but can add drop of serum to promote adhering to glass slide. Procedure: begin with drop of serum. Add drop of Congo red stain. Add organism and emulsify with loop. Second clean slide and draw back. Air dry. Flood slide with maneval’s stain. Blot dry.

Answer 11

Capsule stain

Question 12

A dormant form of the bacterium that allows it to survive poor environmental conditions. Resistant to heat and chemicals because of a tough outer covering made of the protein keratin. For stain, cover with bibulous paper, malachite green, remove paper, rinse with water, counterstain with safranin. Water removes stain from cells but not spores so cells and spores are green

Answer 12

Endospore

Question 13

Used for isolation and differentiation of Staphyloccus aureus which ferments the mannitol and produces acids/lowers pH of the medium resulting in bright yellow colonies with yellow halo. Grows gram positive.

Answer 13

Mannitol Salt Agar.

Question 14

Growth means gram negative. Used to isolate and differentiate members of enterobacteriaceae based on ability to ferment lactose. No growth=organism inhibited by crystal violet/bile; good growth=not inhibited; pink=organism produces acid from lactose fermentation, probable coliform. Colorless= no reaction

Answer 14

MacConkey Agar

Question 15

Growth means gram negative. Used for isolation of fecal coliforms. Inhibit growth of gam positive organisms.

Answer 15

Eosin methylene blue agar

Question 16

Triple sugar iron result interpretation

Answer 16

Yellow slant/yellow butt= glucose and lactose fermentation

Red slant/yellow butt=glucose fermentation, acid production

Red slant/red butt/no butt change= no fermentation

Black precipitate in agar= sulfur reduction

Cracks in agar= gas production

Question 17

How to perform a starch plate analysis. When you add iodine, what does a positive look like?

Answer 17

Divide starch agar plate into 3 sections. Spot inoculate two sections with test organisms. Incubate. Cover growth and surrounding areas with gram iodine. Examine areas surrounding growth for clearing. Usually the growth on the agar prevents contact between the starch and the iodine so no color reaction takes place at that point. But this lack of color change does not mean a positive result-look for a halo around the growth, not on the growth itself.

Question 18

Litmus milk results after incubation

Answer 18

Based on ability to reduce litmus

Lactose fermentation acidifies medium and turns litmus pink.
Reduced litmus is white.
Oxidized litmus is purple.
Acid clots can be dissolved in alkaline conditions.
Fissures in clot indicate gas production.
Stormy fermentation is heavy gas that breaks up the clot.

Pink=acid reaction/lactose fermentation 
Pink/solid, clot not moveable=acid clot
Fissures in clot=gas 
Clot broken apart=stormy fermentation 
White color lower part=reduction of litmus 
Semisolid/not pink, clear/gray fluid at top=curd
Clear medium/loss of body=digestion 
Blue=alkaline reaction

Question 19

What type of hemolysis does S. aureus have? and what do the different types of hemolysis look like?

Answer 19

Beta

There are three main types of hemolysis: beta, fish symbol (alpha), and y.
B=complete destruction of RBCs and hemoglobin-clearing of medium around colonies
Fish=partial destruction, greenish agar around colonies
Y= non-hemolysis; simple growth, no change to medium

Question 20

What is the best antibiotic for treating E. coli?

Answer 20

Penicillin

Question 21

What would a plate look like if the bacteria are resistant to the antibiotic used? (Zone of inhibition)

Answer 21

Size of zone depends on sensitivity of bacteria to the specific microbial agent and the MIC (minimum inhibitory concentration). Bigger zone if antibiotic works, smaller or none if resistant.

Question 22

Mechanisms of antibiotic action

Answer 22

Inhibition of cell wall synthesis 
Inhibition of protein synthesis 
Alteration of cell membranes 
Inhibition of nucleic acid synthesis 
Antimetabolite activity

Question 23

Differential test medium prepared as a base to which a carbohydrate is added. Used to differentiate members of enterobacteriaceae and to distinguish from other gram negative rods. Get 5 of each broth. Inoculate with test organism.

Yellow broth, bubble = fermentation with acid and gas
Yellow broth, no bubble= fermentation with acid, no gas
Red broth, no bubble= no fermentation
Pink broth, no bubble= degradation of peptone; alkaline end products

Answer 23

Phenol Red Broth