Medical Bacteriology Midterm

acid fast stain steps

1. prepare a smear and heat fix

2. smear with carbolfuchsin stain and steam. steam used to force stain into bacteria

3. decolorize with acid-alcohol thoroughly

4. wash with water and counterstain with methylene blue

what is acid-fast stain for

identify mycobacterium

• M.tuberculosis

• M. leprae 

what does positive or negative of acid-fast stain mean

positive

• pink cells

• has mycolic acid in cell envelope

negative

• blue cells

• no mycolic acid in cell envelope

steps of capsule stain

1. add congo red to the slide (colors background)

2. add loopful of bacteria, then drage across slide

3. air dry then add counter stain crystal violet (colors cells)

4. drain, then air dry

what is capsule positive for reference

k. pneumoniae

steps of spore stain

1. prepare a smear and heat fix

2. flood with malachite green and steam. steam used to force dye into bacteria and endospore

3. decolorize with water

4. counterstain with safranin 

what does positive or negative for spore stain mean

positive

• green spore and red bacteria/sporangia

negative

• only red bacteria 

which bacteria can form spores

clostridium and bacillus

gram stain steps

1. prepare smear and heat fix

2. add crystal violet, rinse

3. add iodine, rinse

4. add ethanol, dropwise, rinse

5. add safranin, rinse

which organisms grow in bile-esculin

enterococcus and GDS (S. bovis and S. equinus)

what is in bile esculin agar

• 4% bile salt

• esculin

• ferric citrate

what happens with bile-esculin positive bacteria

1. hydrolyze esculin to form glucose and aglycone esculetin

2. esculetin reacts with iron to form black precipitate

how is bile esculin selective and differential

selective: only some bacteria can grow in 4% bile salt

differential: of the bacteria that can grow, only GDS and enterocci will produce black precipitate 

6.5% NaCl broth

differentiates between GDS and enterococcus

• enterococcus: positive, turbid, bacteria can grow

• GDS: negative, clear, bacteria can’t grow

why differentiate between GDS and enterococcus

enterococcus is more resistant to penicillin

what is PYR test used for

to test for pyrrolidonyl aminopeptidase activity

PYR test steps

1. add bacteria to disk

2. add PYR substrate, then reagent

• PYR positive = red

• PYR negative = no color change

positive PYR

s. pyogenes

negative PYR

s. agalactiae

oxidase test reagent

1% tetramethyl-p-phenylenediamine dihydrochloride

oxidase positive

N. gonorrhoea, pseudomonas

• pink to blue, killed by reagent 

purpose of oxidase test

cytochrome c oxidase activity

oxidase negative

no color change

catalase test reagent

hydrogen peroxide → water and oxygen gas

catalase positive

s. aureus and s. epi

negative catalase

s. pyognes and s.pneumoniae

KOH test on gram positive 

no rxn

KOH test on gram negative

stringy consistency

• weaker cell wall allows cell to be lysed

what is KOH test for

verify gram stain results

what does coagulase do

converts fibrinogen in plasma to form fibrin clot 

what is purpose of coagulase test

differentiate between s. aureus (+) and s. epi (-)

difference between tube test and slide test

tube test - to detect free coagulase

slide test - to detect bound coagulase

blood agar composition

5% sheep’s blood in TSA

alpha hemolysis

partial hemolysis with zones hard to see with the naked eye

• might see green discoloration from oxidation of heme iron

• s. pneumo

beta hemolysis

wide clear zone of hemolysis

• s. pyogenes (GAS), s. agalactiae (GBS), s. aureus 

gamma hemolysis

no hemolysis activity, looks white

• s. bovis (GDS), s. epi

MSA used for

differentiate for mannitol fermentation and select for staph species which can grow in 7.5% NaCl

• s. aureus: mannitol fermentation (+), turns yellow

• s. epi: mannitol fermentation (-), be pink

polar flagellated

one flagellum, one end

lophotrichous

multiple flagella at one end

peritrichous

flagella everywhere

positive semi-solid agar test

entire tube turns red

• bacteria able to reduce TCC while metabolizing

• e.coli, p.aeruginosa

negative semil-solid agar

only area of inoculation turns red

• s. aureus

CAMP test is used for

identify GBS (s. agalactiae)

• produce CAMP factor that increases beta hemolysis of s. aureus (arrow head shape seen when the two bacteria almost meet perpendicularly)

• important to identify since can be fatal to newborns

how is GDS transmitted

fecal contamination of water, contact with infected wounds

MIC

minimum inhibitory conc.

large ZOI

bacteria more susceptible to antibiotic

urine testing medium

I. blood agar

• does not select for anything

II. EMB

• selective (methylene blue): inhibit gram +

• differential (eosin): lactose fermenters will appear purple to shiny green (e. coli). non-lactose fermenters grow as pink

MRSA

appears blue on chromogenic agar

mayo clinic streaking method

1.) streak down the middle

2.) zig-zag across line

3.) rotate 90 degrees and zig-zag again

urine testing numbers

• 0 = no growth

• <1000 = contamination

• 1,000-100,000 = possible infection

• >100,000 = infection

salmonella-shigella

• black precipitate forms with salmonella

what are the reagents for these test:

1.) indole

2.) phenylalanine

3.) decarboxylase (arginine, ornithine, lysine)

4.) catalase

5.) methyl red

6.) Voges Proskauer

1.) Kovac’s reagent

2.) ferric chloride

3.) mineral oil

4.) H2O2

5.) methyl-red indicator

6.) alpha napthol and KOH

TSI slant

• K/A = glucose fermentation

• A/A = lactose/sucrose fermentation

• K/K = no sugar fermentation

• can look for gas and h2s production

Voges Proskauer

determines production of acetoin during glucose fermentation

citrate test

can bacteria do this in the absence of glucose:

citrate → pyruvate (carbon)

ammonium → ammonia (nitrogen)

• pH indicator is bromothymol blue (green → blue)

malonate test

bromothymol blue pH indicator

• green → blue

coagulation test antibody-antigen

phadebact strep b test

RPR

treponema pallidum (syphillis)

ELISA

blue color appears when second antibody (horseradish peroxidase) conjugated to enzyme binds to primary

sterilization - dry heat

material is placed in oven 160 degrees c 1 hour to remove bacteria

autoclaving

15 lbs pressure, 15 min, 121 degrees c

filtration

used to obtain bacterial-free filtrates of heat sensitive solutions

cold sterilization

ethylene oxide (for heat-sensitive objects) and formaldehyde (dry surfaces)

why do we store tests at 4 degree c

slows down reactions and inhibits microbial growth

streak plate method

used to obtain isolated colonies