Med Bac lab final

Ways to sterilize

Dry heat, autoclaving (heat and pressure), filtration, and cold sterielization

The gram stain

Pknk: gram negative, cocci shaped
Purple: Gram positive, rod shaped

Microscopy

Light (the one we use!), dark field, phase contrast, fluorescent, and electron

Numerical aperture of the lens

the measurement of the angle of the maximum cone of light that may enter the objective

What parts of the microscope are used to control illumination? What is the function of the iris diaphragm in a microscope?

The knob for brightness controls the illumination along with the luminous field diaphragm. The iris diaphragm is used for increased focus of the slide

Distinguish the different microscopes

Brightfield: observation of stained, fixed specimens
Dark field: makes the specimen bright against a dark background
PC: for live organisms
Fluorescent: rapid identification
Electron: non-living specimens, scatters specimen with light beams

What temperature for autoclaving? Can all media be autoclaved?

121C. No, some media will denature if autoclaved

Why must blood be added to the agar after autoclaving and cooling? Why si the blood used defimbriated?

Autoclaving will denature the blood nutrients. It is defibrinated so that it can support fastidious bacteria adn inhibit others

Enterococcus can cause

UTIs, wound infections, endocarditis and septicemia

Where are enterococcus found

in the gastrointestinal tract H

how to differentiate enterococcus from streptococci

Enterococci can hydrolyze esculin in the presence of 4% bile salts

All members of enterococcus are

bile esculin positive and grow in 6.5 NaCl broth

Nonenterococci

bile esculin positive but DO NOT grow in 6.5% NaCl broth
more susceptible to antibiotics than enterococci

Describe the cell morphology of enterococci and nonenterococci

Cocci in short pairs or chains

What types of bacteria might be confused with enterococci and nonenterococci in human infections

Streptococcus bovis and Streptococcus equinus

Potentially pathogenic bacteria of the Nose and throat flora

S. pygenes, S. pnuemoniae, S. aureus, C. diptheriae, N. gonnorrhoeae, and N. meningitides

Normal flora of the Nose and throat

nonpathogenic streptococci, staphylococci, Neisseria, and Corynebacterium H

Hemolysis

Alpha: Partial hemolysis of RBC, green decolorization
Beta: Complete hemolysis of RBC, white zone of inhibition
Gamma: no hemolysis, no color change

Preference of blood for BAP

Sheep blood

Coagulase test is used to differentiate

Used to distinguish Staphylcoccus aureus from S. epidermidis

Coagulase test

Extracellular enzyme produced by S. aureus

Bile Solubility can differentiate

S. pneumoniae from other types of a-hemolytic streptococci B

Biel solubility test

A positive result of this test will show dissolution of cells on the media

Oxidase test

Used to see if the bacterium will produce the enzyme cytochrome c oxidase.
Use a wooden spoon NOT a metal loop

Oxidase results

Oxidase positive colonies will begin to turn pink, then dark red, and eventually black in color.

Oxidase positive bacterias

Neisseria and Pseudomonas

CTA sugars

Tests Neisseria species ability to utilize different sugars.
A positive result for this particular test would be the change of pH, by changing it from phenol red to yellow.

CTA sugars

dextrose, sucrose, or maltose

CAMP test

Used for identifying group B streptococcus.
The researcher would look for an increased zone of hemolysis between the streptococci and S. aureus to see results.

CAMP test is incubated under

CO2

Optochin sensitivity and Bacitracin Differentiation

The Optochin sensitivity and Bacitracin differentiation test are used to identify S. pneumoniae and S. pyogenes.
Use of antibiotic disks

Optochin sensitivity

look for a zone inhibition of 14-16 mm in diameter to show a positive result

Bacitracin

any zone of inhibition is considered to be positive for bacitracin

MSA test

used to show if the bacterium utilizes mannitol.

MSA test results

If there is a yellow halo around colonies then that is indicative of mannitol utilization.
CIf a bacteria does not utilize mannitol, but can still survive in the salt concentration, then the medium will produce colonies that do not change to yellow

Catalase test

used to determine if the bacterium has the enzyme catalase
Adding 3% hydrogen peroxide

Catalase is good for differentiating

important for identifying Streptococcus (negative result) from Staphylococcus (positive result)

Catalase test results

Positive result will show immediate bubbling as the hydrogen peroxide is converted to oxygen and water

BAP plates must be incubated in

CO2

How many isolates are taken for the Nose and throat experiment

three isolates
NOT using a fungus

Why should nose and throat cultures be incubated in CO2 and not aerobically

Most bacteriums that reside in the N&T do not need aerobic conditions to thrive

Would you be able to detect Mycobacterium tuberculosis wiht a throat culture? why?

No, TB myst be taken from the liver. But, tuberculosis DNA can be found in an oral swab

Would you find E. coli in a throat culture?

Yes, if one has recently ate or drank something contaminated. Warm conditions are optimal

Why must neisseria be incubated in CTA sugars and not sugar broths?

CTA includes a medium that reduces atmosphere in the media. This reduces the chance of the media getting contaminated

Why should stock cultures be store in slants and not just regualr agar plates?

The slants will not dry out like a plate will

Enzymes that hydrolyze..

starch: amylases
gelatin: gelatinases
Tween-80: lipases
casein: proteases

Bacterias that can hydrolyze large molecules

B. cereus, B. subtilis, B. megaterium (starch)
P. aeruginosa and S. aureus (gelatin)
Mycobacterium smegmatis and M. kansaii (Tween-80)
N. brasiliensis, B. cereus, B. subtilis, and B. megatorium (casein)

Nitrate reduction

Reduction of nitrates to nitrites and nitrogen gas

Almost all enterobaccerium can....

Reduce nitrate

Neisseria mucosa

reduce nitrates

Neisseria overall cannot

reduce nitrate

Nitrate reduction results

development of red color means that it has been reduced
if no red color develops, it is possible that the test further reduced into other compounds

Zinc reduction test

Can be used to see if there is still nitrate present in broth

Zinc reduction results

red color means that there is nitrate still present in broth
no red color means that nitrate has been fully developed

Starch agar plate

after incubation, flood the plate with Grams Iodine

Starch agar plate results

positive results will have clear areas around colonies meaning that starch was hydrolyzed
Negative results will be dark-blue

Casein agar plate

Look for clear zones around colonies

Gelatin tubes test

look for liquification

Tween 80 test

positive test will turn from yellow to pink

Three groups of stains

acid, basic, and neutral

Spore stains

Dorner spore stain and Malachite Green spore stain

Dorner spore stain

use of carbolfuchsin and steam. Covered with nigrosin.
Spores should be red, sporangia colorless and background gray or black

Malachite spore stain

use of malachite and steam. Counterstain wiht safranin
Spores should be green in red sporangia

Acid-fast stain

Use of carbolfusion and steam. Decolorize to a faint pink color with acid-alcohol. Counterstain with methylene blue.
Acid-fast organisms will appear pink to red while non will be stained blue

Nigrosin stain

use of nigrosin drops
cells are unstained against dark background

Congo red stain

use of 2% congo red stain drops. flood smear with acid-alcohol
cells are unstained against dark background C

capsule stain

encapsulated cells are grown in skin milk and stained wiht crystal violet and copper sulfate solution
cells appear faint blue/clear on a purple background
NO HEAT FIXING

Motility determination in semi-solid agar

motility is indicated by the appearance of a red diffuse zone of growth
A negative result will show red growth only along line of innculation

Simple mount

used for seeing if the live organisms is moving
will be necessary to reduce illumination to see movement

Hanging drop

use of vaseline as buffer B

Brownian movement

Movement from the cover slip and liquid interacting

Flagella stain

young cultures are generally used
Mordant, tannic acid is used to coat and thicken the flagella

Minimal inhibitory concentration

MIC
defines in vitro levels of susceptibility or resistance of specific bacterial strains to applied antibiotic.

Most agents used will be resistant if the zone of inhibition is...

14 or less

Most agent used will be susceptible if the ZOI is...

18 or more

Why is Mueller-Hinton agar used instead of typtic or nutrient agar?

IT is a loose agar so it is easier for the diffusion of the antibiotic plates

Presumptive diagnosis of gonorrhea

Gram negative diplococci
oxidase positive

N. gonorrhoeae must be incubated in

CO2

Common method used to enumerate urine specimen

calibrated loop direct streak method on BAP and MAC/EMB

Urine bacteria count meanings

less than 1000: suggestive of contamination
between 1000 and 100,000: possible infection
more than 100,000: indicative of infection
Remember that the plate must be multiplied by 100L

List some factors that might give artifically low or high urine counts

Low pH, diet, hydration, and exercise

Bacteria found in stool flora

Enterobacteria, gram positive cocci, yeasts, clostridia, and bacteroides.

Media used to differentiate Stool bacteriums

EMB, MAC, and leifson deoxycholate agar
All of these inhibit gram positive bacteria and contain lactose

TSI is used for

differentiation of bacteria in the family Enterobacteriaceae

TSI components

glucose, lactose and sucrose

TSI results

Alkaline slant, acid butt: Glucose fermented
Acid slant, acid butt: lactose and/or sucrose ferm.
Alkaline slant, alkaline butt: None of the sugars ferm.
Gas bubbles in butt: Aerogenic culture
Blackening of medium: Hydrogen sulfide produced

GasPak system

allows for growth of anaerobes

Why is ferrous acid needed to detect H2S in TSI agar?

It is needed to produce iron ions W

What is the purpose of vitamin K1 in Brucella agar?

Supports the growth of certain strict anaerobes

IMViC reactions

Indole, Methyl red, Voged-proskauer, and Citrate

Indole test

tube of tryptophan broth, add Kovacs reagent
development of red color indicated indole prod.

Methyl red

MRVP medium (has phosphate, polypeptone, and dextrose)
add 5 drops of methyl red indicator
red color is positive (ability to maintain acid pH), negative are yellow

Voges-Proskauer

MRVP medium, add 5% a-naohtol in absolute ethanol and 40% KOH
development of red color is positive for acetylmethylcarbinol prod.

Citrate test

Simmons citrate agar
Blue color is positive (bacteria can grow on medium wiht citrate as sole carbon source and ammonium salts as nitrogen source)U

Urease broth

Tube of urease broth. Incubated in water bath
red color indicates strong urease activity

Strong urease activity is characterisitc of

Proteus

Phenylalanine agar

add drops of 10% ferric chloride
formation of green color on slant is positive of deamniation of phenyl. to phenyl pyruvic acid

Decarboxylation test

Lysine, Arginine, and Ornithine broths. Overlay with mineral oil
Color change to violet wiht growth is positive and presence of enzymes

Malonate Broth

Change in color from green to Prussian blue is positive for malonate utilization

Glucose broth

two tubes, one overlayed with oil
change in color of aerobic tube is sugar utilization whereas change in oil tube is sugar fermentation