CBU Bio205L MicroBio Lab Practicum 2

Staphylococcus

• gram positive spherical organisms about 1mm

• occur singly in pairs, irregular clusters, salt tolerant

what does staphylococcus aureus cause

food poisoning, toxic shock syndrome, boils, and carbuncles

what does staphylococcus epidermidis cause

saprobe of skin; rarely cause human infection

what does staphylococcus saprophyticus cause

UTI

isolation of staphylococci purpose

to isolate and identify staphylococcal species from nasal cavity and other environments

s.aureus characteristics

• medium/large, raised colonies on blood agar, cream/golden yellow pigment on colistin nalidixic acid agar

• gram positive cocci in clusters

• beta-hemolytic on sheep blood agar

• bound coagulase (clumping factor) = positive

• free coagulase = positive

• MSA = growth and fermentation

• DNase = postive

mannitol salt agar

medium that is both selective and differential

phenol red

• indicator of MSA to detect acid production

• change from red to yellow in presence of acid producing s.aureus

ex 44 procedure step 4

• observe plates or presence of round opaque colonies that are white/pigmented (staphylococcus colonies)

• s.aureus produces golden yellow, others produce white

• colonies surrounded by yellow halo contain bacteria fermented mannitol

• s.epidermidis/saprophyticus micrococcus luteus don’t ferment mannitol

ex 44 procedure step 5

• typical clusters of gram positive cocci should appear

• micrococcus luteus could be isolated on medium; are gram+ cocci but characteristics are tetrads (clusters of four) or cuboidal packets of 8 (sarcina configuration). bright yellow colonies on agar

members of genus staphylococcus

• found in humans, other animals, and the environment

• on humans, they can be found on skin/nasal cavity leading to infection

s.aureus biochemical characteristics

• produce coagulase, enzyme that clots blood plasma/enhances its virulence by providing resistance to phagocytosis

• produces DNase to digest DNA

• sensitive to antibiotic novobiocin

which staphylococcal is resistant to novobiocin

s.saprophyticus

staphylococcal species purpose

to distinguish between species of staphylococcus based on biochemical characteristics

characteristics of s.aureus

color: golden yellow

mannitol acid production: yes

coagulase: yes

DNase: yes

novobiocin sensitive: yes

characteristics of s.epidermidis

color: white

mannitol acid production: no

coagulase: no

DNase: no

novobiocin sensitive: yes

characteristics of s.saprophyticus

color: white

mannitol acid production: no

coagulase: no

DNase: no

novobiocin sensitive: no

coagulase activity

• use coagulase plasma

• determined by tilting each coagulase tube to side and noting the presence of a solid immovable clot, if plasma still flows clot formation of coagulase has not take place

dna digestion

• use DNase test agar plate

• test for dna digestion by flooding plate w/ 1N hydrochloric acid

• dna will normally react w/ HCl and form a very fine precipitate which gives cloudy appearance to agar

• if dna digestion has occured the area near the streak will remain clear, remainder of plate becomes cloudy

novobiocin sensitivity

• use mueller-hinton agar and make bacterial lawn

• determined by measuring diameter of halo (zone of inhibition) around disc where no bacterial growth has occurred

• if zone< 17mm bacterium is resistant, if zone> 17mm bacterium is sensitive

enteric bacteria

• gram- nonspore-forming rods that normally inhabit animal/human intestinal tract

• belong to family enterobacteriaceae

• facultatively anaerobic and can cause typhoid fever, shigellosis, and infantile/traveler’s diarrheas

enteric bacteria purpose

to isolate and identify enteric bacteria on selective and differential media

how enteric bacteria is isolated

cultivating fecal samples on differential media containing lactose

MacConkey agar

• differential medium containing bile salts to inhibit nonenteric bacteria

• has 2 dyes: neutral red and crystal violet which are taken up by lactose-fermenting bacteria

• crystal violet inhibitory to growth of gram+ bacteria

• enteric bacteria for pigmented colonies while nonlactose fermenters form colorless

• lactose fermenting bacteria will appear in red colonies

• escherichia coli brick red, enterobacter, klebsiella, pink-red mucoid texture

EMB (eosin methylene blue) agar

• another differential medium that does not support growth of gram+

• pigmented colonies formed by lactose fermenters, but type of pigmentation is distinctive to various genera

• pigmented colonies that are black-blue w/ green metallic sheen in reflected light are probably escherichia coli

• enterobacter aerogenes convex/mucoid w/ dark centers and creamy pink borders

• nonlactose fermenters will yield colorless colonies

triple sugar iron agar (TSI)

• medium for differentiating enteric bacteria

• contains glucose, sucrose, and lactose and pH indicator phenol red

• bacteria inoculated into slant of medium and deep portion/buttt where anaerobic conditions prevail

bacteria on tsi agar purpose

to differentiate enteric bacteria based on acid and gas production

three reactions in TSI agar

• species that only ferment glucose (salmonella): both slant and but will turn yellow, but then butt stays yellow slant goes back to red from alkaline conditions reappearing from digestion of peptones and production of ammonium compounds

• if glucose, lactose, and/or sucrose are fermented, sufficient acid will be produced to change and keep slant/butt yellow

• if no carb fermented, slant and but stays red

gas production from carbs

• presence of cracks/fissures in medium after incubation

• large amounts of gas produced may push medium up tube

hydrogen sulfide production in TSI agar

• tsi agar contains iron ions and sodium thiosulfate

• certain bacteria use thiosulfate in their metabolism and release hydrogen sulfide, reacting with iron making iron sulfide which turns butt black

characteristics of escherichia

lactose fermentation: +

slant: acid butt: acid

gas: + H2S: - I: +

+acidacid+-+

characteristics of klebsiella

lactose fermentation: +

slant: acid butt: acid

gas: + H2S: - I: ±

characteristics of serratia

lactose fermentation: -

slant: acid butt: acid

gas: - H2S: - I: -

characteristics of proteus

lactose fermentation: -

slant: alk butt: acid

gas: ± H2S: ± I: ±

characteristics of pseudomonas

lactose fermentation: -

slant: alk butt: alk

gas: - H2S: - I: -

IMViC series of four tests

indole production (I), methyl red test (M), voges-proskauer test (V), citrate test (C). “i” in acronym for pronunciation

indole test

identifies species that can digest tryptophan to indole and other products

• 5 drops of kovac’s reagent to tryptone broth/trypticase

• red surface means indicates indole production

methyl red test

depends on organism’s’ ability to ferment glucose and produce large amounts of acid. added at end of incubation period, remains red in acid solution and turns yellow in alkaline/neutral solution

• 5 drop methyl red to one MR-VP tube

• red means positive for acid production, disappeared means negative

Voges-proskauer test

uses digestion of glucose to acetylmethylcarbinol to differentiate between enteric species. if present acetylmethylcarbinol will react w/ alpha naphthol and potassium hydroxide to form a red chemical compound

• 10 drops Barritt’s A reagent to VP broth tube

• red means positive, lack of color means negative

citrate test

based on ability of certain bacteria to use citrate, a salt of citric acid as a sole carbon/energy source. pH of medium rises and indicator bromothymol blue becomes deep blue. if not citrate occurs original color green remains

• note presence of growth on Simmons citrate agar

IMViC series purpose

to further differentiate enteric bacteria based on additional biochemical tests

some characteristics of escherichia coli

temp: 37c

morphology: rod

arrangement: single

gram reaction: -

lactose fermentation: AG

H2S production: -

indole production: +

some characteristics of bacillus subtilis

temp: 37c

morphology: rod

arrangement: single

gram reaction: +

lactose fermentation: -

H2S production: -

indole production: -

some characteristics of micrococcus luteus

temp: 25c

morphology: coccus

arrangement: packet

gram reaction: +

lactose fermentation: -

H2S production: -

indole production: -

some characteristics of serratia marcescens

temp: 25c

morphology: rod

arrangement: single

gram reaction: -

lactose fermentation: -

H2S production: -

indole production: -

genus bacillus species

• over three dozen species, significance in industrial/medical microbiology

• dried b.subtilis used a source of enzymes in enzyme detergents

• b. polymyxa produces antibiotic polymyxin

• geobacillus stearothermophilus used to test effectiveness of sterilization

• b.anthracis agent of anthrax, serious disease of animals and health threat to humans

genus bacillus characteristics

• gram+ sporeforming rods that produce catalase

• most are motile, and many species digest starch and DNA

• other soilborne aerobic organisms will be destroyed with heat to select out bacillus spores

genus bacillus purpose

isolate and identify bacillus species from soil samples

genus bacillus steps

• heat water bath to 80c

• place generous pinch soil in water in test tube, immerse in water bath 10 mins, remove let cool

• destroys most organisms and will stimulate spores to germinate

• prep nutrient agar and starch agar plate

catalase test

placing drops of hydrogen peroxide onto colonies on nutrient agar and note the formation of bubbles of oxygen gas

starch test

many bacillus produce enzyme amylase that digests starch. by adding iodine to colonies growing on starch agar, amylase will be produced if agar around colonies are digested and there’s nothing for iodine to react to, resulting in clear area around colonies. black around colonies means starch wasn’t digested so no amylase produced

effect of antibiotics on bacteria purpose

to evaluate the sensitivity of bacterial species to antibiotics

disk susceptibility test/kirby-bauer method

bacteria is streaked on mueller-hinton agar which gives reproducible results and doesn’t inhibit sulfonamides.

zones of inhibition

paper discs containing known amounts of the antibiotic are added and plates are observed for clear zones