Lab Practical 2

What does “anaerobic” mean?

without oxygen

What are we going to use fluid thioglycollate medium to indicate?

the presence/absence of oxygen

How will we make a jar anaerobic?

we place a hydrogen generator packet in the jar, along with a catalyst. The hydrogen reacts with the oxygen in the car.

Define each of the following and state the location of the most growth for each in thioglycollate medium: obligate anaerobe, obligate aerobe, facultative anaerobe, aerotolerant anaerobe, microaerophile

• obligate anaerobe: cannot grow when O2 is present, grows in the bottom half of the tube

• obligate aerobe: grows only when O2 is available, grows within the red zone at the top of the tube

• facultative anaerobe: grows best when O2 is available, but also grows without it, growth all the way through the tube with the most growth within the red zone at the top of the tube

• aerotolerant anaerobe: grows equally well with or without O2, even growth all the way through the tube

• microaerophile: grows only if small amounts of O2 are available, just below the red zone near the top of the tube

What does the oxidase test tell us about bacteria?  Describe what a positive and negative result looks like.

it tells us whether or not the bacteria make oxidase, an enzyme essential for aerobic respiration.

Positive test (oxidase positive) - the reagent turns blue/purple within 15 seconds

Negative test (oxidase negative) - yellow or no color within 15 seconds

catalase breaks down ___(1)_____, producing ____(2)_____ and ____(3)_____.

(1) hydrogen peroxide

(2) oxygen

(3) water

how can one tell if catalase is acting on its substrate?

bubbles

phenol red broth is a differential media. what does a color change in a phenol red broth indicate about a bacterium that has been growing in it?

it indicates whether or not the bacterium has enzymes necessary to ferment the sugar in the tube

Phenol red broth contains a pH indicator called phenol red, which indicates a pH change when it changes color. State the color of the broth under the following conditions:

Neutral pH ____

Acidic pH ____

Alkaline pH ____

Neutral pH = red

Acidic pH = yellow

Alkaline pH = pink

what is a Durham tube? what does it collect?

a tiny tube that sits upside down in the phenol broth tubes. It collects gas

what two products may be produced during fermentation of simple carbohydrates? how is each identified in the fermentation test? can you have one without the other?

acid and gas; acid turns phenol red, yellow; gas collects in the Durham tube so you can see a bubble there; yes, you can have one without the other

name 3 simple carbohydrates that can be added to media for biochemical testing

glucose, sucrose, lactose

label the oxygen requirements for each thioglycolate medium

which is oxidase positive and oxidase negative

which is catalase positive and catalase negative

what are the results of each tube

A - turned yellow, so it ferments sucrose, but there is no gas bubble so it produces acid only

B - stayed red, no gas bubble, does not ferment sucrose

C - turned yellow and there is a gas bubble, so it ferments sucrose with both acid and gas production

disinfectant

chemical agent used on inanimate objects to lower the level or number of microbes on the surface

antiseptic

chemicals used on living tissue to decrease the number of microbes

antimicrobial agent

a general term for drugs, chemicals, or other substances that either kill or slow the growth of microbes

what is an antibiotic

a drug used to treat bacterial infections

what is a narrow spectrum antibiotic

an antibiotic effective against a restricted number of bacteria

ex: penicillin G, amoxicillin

what is a broad spectrum antibiotic

an antibiotic effective against a wide range of both gram-positive and gram-negative bacteria

-cidal

to kill

static

to inhibit

a disinfectant must _________ the pathogen while in contact with them

kill

list two types of bacterial cells that are more resistant to disinfectants

1. endospore-formers (bacillus, clostridium)

2. acid-fast bacteria (mycobacterium)

why is antimicrobial susceptibility testing done

to determine which antibiotics will be most effective against a specific infection

what is the name of the antimicrobial susceptibility test also known as the filter paper disk agar diffusion method

the kirby-bauer test

what is the filter paper disks of the kirby-bauer test

antibiotic

what kind of agar is used in the kirby-bauer test

mueller-hinton agar

what is streaked onto the agar plate in the kirby-bauer test

a lawn of bacteria

where are the filter paper disks placed in the kirby-bauer test

on top of the bacteria on the agar plate

what happens to the antimicrobial agent in the disk after it contacts the agar in the kirby-bauer test

it diffuses away from the disk in all directions

after incubation, what does one look for on the plates in the kirby-bauer test

zone of inhibition around the disk

what does R mean interpretation? how is it determined

• Resistant

• compare the width of the zone of inhibition to the standardized table of antibiotics

what does S mean interpretation? how is it determined

• Susceptible

• compare the width of the zone of inhibition to the standardized table of antibiotics

name a broad spectrum antibiotic

ampicillin

name a narrow spectrum antibiotic

penicillin G 

amoxicillin

what is enriched media? give examples

allows many types of bacteria to grow - even fastidious bacteria

ex: blood agar, chocolate agar

what is differential media? give examples

allow you to distinguish one group of bacteria from another after they have grown on the media

ex: mannitol salt agar, blood agar

what is selective media? give examples

allow some types of bacteria to grow and not others

ex: mannitol salt agar

describe the characteristics of staphylococci

gram-positive spheres growing in irregular clusters; catalase-positive; salt-tolerant

where is S. epidermidis normally found? does it cause any diseases? if so, what?

skin and mucous membranes

it doesn’t normally cause any disease

where is S. saprophyticus normally found? does it cause any diseases? if so, what?

it is found only infrequently on our bodies

it is associated with urinary tract infections in women from ages 16 -21

where is S. aureus normally found? does it cause any diseases? if so, what?

skin, internal nares, vagina

diseases - TSS (toxic shock syndrome), food poisoning, scalded skin syndrome, pneumonia, meningitis, sinusitis, otitis media, abscesses

what does hemolysis do

destroy red blood cells

what does coagulase do

coagulate plasma

what does leucocidin do?

destroys white blood cells

what does hyaluronidase do?

destroys connective tissue (“spreading factor”)

what does staphylokinase do?

dissolves fibrin clots

what does enterotoxin do?

affects the digestive system (produced in food, not the body)

what staph species produces hemolysins, coagulase, gelatinase, leucocidin, hyaluronidase, staphylokinase, and enterotoxin?

staphylococcus aureus

what does mannitol salt agar (msa) select for? what ingredient is present in msa that causes it to be selective?

msa selects for salt-tolerant bacteria (like staph)

9.5% salt

how is msa differential? what does it differentiate between? what ingredients are present in msa that cause it to be differential?

it distinguishes mannitol fermenters form mannitol nonfermenters

msa contains mannitol and an acid-base indicator

s. aureus colony color on blood agar

yellow

s. epidermis colony color on blood agar

white

s. saprophyticus colony color on blood agar

white or yellow

s. aureus type of hemolysis on blood agar

beta

s. epidermidis type of hemolysis on blood agar

gamma

s. saprophyticus type of hemolysis on blood agar

gamma (usually)

s. aureus coagulase (+ or -)

+ (forms a clot)

s. epidermidis coagulase (+ or -)

-

s. saprophyticus coagulase (+ or -)

-

s. aureus mannitol fermentation (+ or -)

+

s. epidermidis mannitol fermentation (+ or -)

-

s. saprophyticus mannitol fermentation (+ or -)

varies +/-

s. aureus novobiocin (S or R)

S

s. epidermidis novobiocin (S or R)

S

s. saprophyticus novobiocin (S or R)

R

describe the members of the genus streptococcus

gram-positive spheres arranged in chains; catalase negative

what does fastidious mean?

these bacteria need enriched medium to grow on

what type of media do strep need to grow on?

enriched (blood agar plates)

substances that destroy red blood cells are called _______

hemolysins

one way strep are classified is by the type of hemolysis they produce on blood agar. describe the following:

• alpha hemolytic strep

• beta hemolytic strep

• gamma strep

• alpha hemolytic strep: BAP turns red to green because they incompletely break down hemoglobin

• beta hemolytic strep: BAP clears to colorless because they completely break down hemoglobin

• gamma strep: no change

which group causes disease? list several of the diseases that are caused by a member of this group

beta hemolytic (streptococcus pyogenes)

diseases caused include toxic shock syndrome, strep throat, scarlet fever, and rheumatic fever

a second way strep can be classified is by the type of carbohydrate antigens present in their cell wall. what is an antigen?

any substance that causes antibodies to be produced by the body; it reacts only with its specific antibody

how are these strep named?

Group A, Group B, ….. Group T

Human pathogens belong to which group?

Group A

describe the Enterobacteriaceae

gram-negative nonsporing rods, facultative anaerobes

name three species of the Enterobacteriaceae that are pathogens. name the disease that each causes

1. klebsiella pneumoniae - causes pneumonia

2. shigella dysenteriae - causes shigellosis (bacillary dysentery)

3. salmonella typhi - causes typhoid fever

how do these pathogens differ from the non-pathogenic forms?

the pathogens do not ferment lactose, but the non-pathogenic forms do ferment lactose

do all E. coli normally cause disease?

no

what are MacConkey and EMB agar selective for

gram-negative rods

what do MacConkey and EMB agar differentiate?

lactose fermenters from non-lactose fermenters

phenylalanine is broken down into ______________ when bacteria that produce phenylalanine deaminase are grown in culture

phenylpyruvic acid

in the phenylalanine deaminase test, a reagent called ferric chloride is added after incubation. what does a positive reaction look like (color)?

green

name a genus in Enterobacteriaceae that is phenylalanine deaminase positive

proteus

urea is hydrolyzed into ______ and _______

carbon dioxide and ammonia

name the exoenzyme studied in the urea hydrolysis exercise

urease

name one genus of bacteria that can hydrolyze urea

proteus

after incubation, how can you tell if a urea slant was inoculated with urease-positive organism or a urease-negative organism?

urease-positive organism turned urea slant pink

urease-negative organism didn’t 

name the indicator that was added to the urea slant before inoculation

phenol red

this indicator is ______ in a solution below pH 8.4 and _______ above pH 8.4

yellow/orange, red/pink

we will be working with “SIM” medium. In the acronym what does each letter stand for

S stand for sulfide

I stands for indole

M stands for motility

name the enzyme responsible for sulfide production

cysteine desulfurase or thiosulfate reductase

after incubation, how can you tell if a SIM tube has been inoculated with an organism positive for sulfide production?

the SIM medium turns black 

Name a genus in Enterobacteriaceae that is positive for sulfide production

salmonella, shigella, proteus

In the presence of tryptophanase, tryptophan breaks down into _______________ plus ammonia.

indole

What does Kovac’s reagent indicate?

the presence of indole

After incubation, how can you tell if a SIM tube had been inoculated with an indole-positive organism or an indole-negative organism?

if you get a red color after adding Kovac’s reagent, its indole-positive