Microbio Lab Practical Pt 2 10-15

Gram Positive Cocci Study Day 1

Catalase test, determine arrangement
+ Cat, clusters= staphylococcus

- Catalase, chains= streptococcus

Gram Positive Cocci Study Day 2 Staphylococcus

Mannitol Salt Agar: Selective and differential medium

Inhibitor:7.5% NaCl selects for Staphylococcus

Indicator: Phenol red

Contains 1% Weight/Volume of D-mannitol, sugar only fermentable by Staph Aureus/Saprophyticus

Day 2 Positive

Agar plate turns yellow, d-mannitol fermenters are present means staph aureus or saprophyticus are present

Day 2 Negative

Agar plate remains red, mean that organism cannot ferment d-mannitol, indicates presence of Staph epidermidis

Gram positive cocci study Day 2 Streptococcus

Blood Agar plate is used to differentiate streptococcus bacteria based on hemolysis patterns
Produce small pinpoint gray colonies

Day 2 alpha hemolysis

Strep pneumoniae exhibits “average” hemolysis patterns

Day 2 Beta Hemolysis Pattern Strep

Either Strep agalactiae or Strep pyogenes produce Beta hemolysis patterns, require further differentiation

Gram positive cocci differentiation Step 3 Strep

Bacitracin (antibiotic) sensitivity test

Gram positive Bacitracin sensitive

Strep. pyogenes, larger zone of inhibition

Gram positive bacitracin resistant

Strep. agalactiae, smaller zone of inhibition

Step 3 gram positive differentiation staph

Coagulase: inoculate a tube containing rabbit plasma

DNase: inoculate a plate containing DNA bound to methyl green indicator

Positive Coagulase

A positive result indicates the species possesses coagulase, an enzyme that forms of fibrin clots in plasma

Fibrin clots protects bacteria from host

Positive, the plasma will gel in tube

S. Aureus contains coagulase

Positive DNase

A positive DNase test is associated with a clearing of blue pigment associated with DNA presence around the colony

S. aureus contains DNase

Enterobacteriaceae common features

Gram negative rods

ALL ferment glucose

ALL nitrate positive

ALL oxidase negative

mostly motile

Primary Pathogens

Organism causes disease in anyone

Opportunistic pathogens

Cause diseases under certain conditions in certain hosts

Gram negative study bacteria unknown composition

E. Coli/Enterobacter aerogenes + Salmonella/Proteus mirabilis

Day 1 Isolation

Streak mixed culture on EMB, MAC, Salmonella Shigella agar, and Bismuth Sulfite agar

MAC Components

MacConkey Agar
Crystal Violet and bile salts inhibit Gram positives

Neutral red detects lactose fermentation

MAC results

• GRowth w pink color: gram (-) and lactose (+)

• Growth w/ out pink color: Gram (-) and lactose (-)

EMB Components

Eosin & Methylene blue inhibit gram positives

Eosin and methylene blue detect lactose fermentation

EMB Results

• Growth w/ pink to lavender color: Gram (-) and weak lactose fermentation (+)

• Growth w/ dark purple or green sheen: Gram (-) and strong lactose fermentation (+)

• Growth w/out color change: Gram (-) and lactose (-)

Salmonella-Shigella Agar Components

• Brilliant green, bile salts, and sodium citrate inhibit Gram positives and many gram negatives

• Neutral red detects lactose fermentation and iron indicates sulfur reduction

Salmonella-Shigella Agar Results

• Growth w/ pink to red color: gram (-), lactose (+), sulfur (-)

• Growth w/out pink color change: Gram (-), lactose (-), sulfur (-)

• Growth w/ black precipitate: Gram (-), lactose (-), sulfur (+)

Bismuth Sulfite Agar Components

Bismuth sulfite and brilliant green dye inhibit Gram positives and many gram negatives

Iron and bismuth sulfite detect sulfur reduction

Bismuth Sulfite Results

Growth w/out black precipitate: gram (-) and sulfur (-)

Growth w/black precipitate: Gram (-) and sulfur (+)

Day 3 Confirmatory gram negative

Differentiation of E. Coli from E. aerogenes

Salmonella from P. mirabilis

Day 3 gram negative E. coli from E. aerogenes

IMViC

E. coli: Indole, MR++

E. aerogenes: VP, Citrate ++

Day 3 gram negative Salmonella from proteus mirabilis

Salmonella: SIM+, Urea-

Proteus mirabilis: SIM+, Urea+

Tryptone broth

Detects tryptophanase: hydrolyzes tryptophan→indole, pyruvate and ammonia (NH3)

Add 3-5 drops of Kovac’s reagent

+:cerise/red

-: no change

Methyl Red (MR)

tests for mixed acid fermenters

add 3-4 drops of methyl red

(+) reagent remains red

(-) red color is not retained

Vogues Proskauer

Tests for 2,3-butanediol fermenters

1. Add 10 drops of VP I (alpha naphthol)

2. Add 10 drops of VP I (KOH)

3. (+) red color

4. (-) no red color

Simmons citrate

Detects utilization of citrate as sole carbon source for growth

Indicator: bromothymol blue

(+) growth, possibly accompanied by blue color

(-) no growth

green→blue

SIM

Used to determine sulfur reduction, indole production, and motility

Sulfide: H₂S produced by bacterium reacts with Fe in medium to produce FeS, (+)=black precipitate

SIM

Indole

SIM

Motility

+=cloudy

Urea broth

Detects enzyme urease, degrades urea→2 ammonia (NH₃) and carbon dioxide (CO₂)

Indicator:Phenol red

pH increase over 8.1: Cerise

Neutral pH→red

pH decrease→ yellow

Positive=cerise color (alkaline)

Negative= yellow for acidic, red for neutral

-Cide/-Cidal

Kill microbes

-Static

Inhibits microbial growth

Disinfectants

Strong chemical agent used on inanimate surface

Not as effective as sterilization, may not kill resistant spores

Antiseptics

Chemical agents that may be safely applied to living tissue

Kill or inhibit microbes and prevent infection

Antibiotics

Compounds that may be administered for systemic circulation (pills, injections, other methods) for the purpose of killing bacteria/inhibiting growth

Most synthetic analogs of naturally occurring compounds

Kirby Bauer

Standard antibiotic sensitivity test

Antibiotic impregnated disc placed on a lawn of bacteria prior to incubation

Standardized to ensure correct interpretation

Zone of Inhibition

Zone is measured to determine if an organism is susceptible, resistant, or intermediate to a certain antibiotic

Kirby Bauer Agar

Mueller-Hinton Agar

pH: narrow range, 7.2-7.4, pH outside of this range affects stability and function of antimicrobials

Nutrient composition of some media have factors that can interfere with antimicrobial function

Depth of agar- 4mm, can affect lateral diffusion rate of antibiotics

Too low: overestimation of sensitivity

Too high: swamp antimicrobial or quickly deplete nutrients, affects growth

When is kirby-bauer performed?

To identify best antibiotic or combination for treating infection

Under extraordinary circumstance: infectious agent unknown, antibiotic resistant, or life threatening condition

Diffusion area

The area across which antibiotic diffuses from the disk

Concentration is highest near disk and decrease

Kirby Bauer

1) Mueller hinton agar

2) Create solid lawn of bacteria with swab

3)Streak in one direction, rotate 90 degrees, sweep in other, rim the plate

4) Label plates with culture

5) discs applied with applicator

Environmental Condition Requirements of all organisms

Temperature

Osmotic pressure

pH

UV-radiation

Oxygen

Cardinal temperatures

Every organism has a Minimum, optimum, and maximum

Optimum tends to be closer to maximum

Temperature test

Tubes incubated at varying temperatures, determination made based on turbidity

Geobacillus is a thermophile

E. coli is a mesophile

Serratia marcescens

Produces secondary metabolite prodigiosin

Produced at 25^o but not 37 degrees

TSA slants

Acidophiles

Prefer low pH

Neutrophiles

Prefer pH 7

Alkaliphile

Prefer high pH

pH optima or range determination

With Nutrient Agar plates of varying pHs (5,7,9)

Determined by amount of growth

Osmotic pressure tolerance

Tolerance to solutes in environment (normally salinity)

Nonhalophile

Do not tolerate salinity

Halotolerant

Tolerate salinity

Halophile

Salt lovers

Oceans

Halobacterium

Extreme Halophile

Extreme salinity

Great salt lakes

Effects of UV radiation

Absorbed by DNA, can destroy DNA and kill bacteria

Causes formation of pyrimidine dimers (thymine dimers)

DNA damage prevents replication

Disinfects

UV Test

Irradiate for prescribed time

Incubate at 37

Be careful, can burn eyes

Obligate aerobe

strictly requires oxygen

Obligate anaerobe

Strictly requires ABSENCE of oxygen

Facultative/aerotolerant anaerobes

Can tolerate and grow in both

Microaerophile

Require oxygen at lower than atmospheric concentrations

Fluid Thioglycolate Broth

Contains thioglycolate, reduces amount of oxygen present, and small amount of agar.

Creates oxygen gradient

Top of tube is oxygen rich, bottom is anaerobic

Resazurin added to indicate presence of O₂

37 degree incubation

Water testing reasons

Routinely test for coliform bacteria

Indicates fecal contamination of water

Coliform bacteria

Members of Enterobacteriaceae

Gram negative rods

ferment lactose with gas production

Found in feces

Day 1 Presumptive Test Water Testing

Multiple tube lactose fermentation

• Identifies lactose fermentation with gas production

• Used as initial screening for presence of coliform bacteria

Does not conclusively indicate fecal contam, suggestive

MPN Method

Estimates number of microbes present

Sample diluted into lactose broth at double or single strength

• 3 tubes with 10 mL of sample (2X LActose)

• 3 tubes with 1 mL of sample (1X Lactose)

• 3 tubes with 0.1 mL of sample (1X lactose)

Contain durham tubes

Day 2: Confirmed Test

Loopful of culture from a positive lactose tube to perform a 3-zone streak on EMB or Endo Agar

Day 3 Completed Test

Observe:

EMB: Selective for gram (-) and differential for lactose fermenters (often fecal coliforms)

Endo Agar: selective for gram (-) and differential for lactose fermenters (often fecal coliforms) E. coli produces pink/red colonies, metallic sheen

Then gram stain and inocculate lactose broth to confirm

Membrane Filter Technique

Passes contaminated water across a filter with 0.45 um pores

Captures bacteria, allows water/viruses to pass

Filter then placed in differential medium to identify coliform presence

mENDO Agar (pink) shows total coliforms by golden sheen

mFc Agar (light blue) shows fecal coliforms as darker blue colonies on the agar