Chloe Evetts (8990-4674)
May 20, 2013
DIAGNOSTIC MIROBIOLOGY – Culture and isolation of microbes
CULTURE MEDIAAND BIOCHEMICAL TESTS
An individual species has it’s own distinct “fingerprint”; It will produce certain results to different
tests, usually resulting in a combination of outcomes unique to that individual species. These
difference arise from the fact each microbe has adapted to it’s own micro-environment, and
since each micro-environment, for all intents and purposes, is unique, it has developed certain
sensitives and resistances to each variable found.
The purpose of this lab is to utilize the various biochemical techniques and selective/differential
media in order to analyze and determine an unknown species. The reactivity of our species to
each test will be recorded which will help us learn how to use the Gideon Database to identify a
By running our unknown through various biochemical tests and by plating on selective and/or
differential media coupled with proper aseptic techniques, we should end up with a unique set of
results that can matched to an online global infectious disease database, Gideon, to identify our
Because the species were originally cultured in the classroom, one can assume species that
thrive in low pH, extremely low and extremely high temperatures, and that are
anaerobic will not be found. Instead, bacteria commonly found on the hands, mouth,
and ground will more than likely be discovered.
1 Selective and Differential Media
a Each plate will be divided up with a sharpie into 4 sections
b After plating, incubate plates for 24-48 hours
c Blood agar plates
i Observation of green discoloration of the medium= alpha hemolysis
ii Clearing of the medium/complete lysis = beta hemolysis
iii + control alpha hemolysis: E. coli
iv + control for beta hemolysis: Strep. pyogenes
d MacConkey Agar
i Gram positive bacteria will not grow ii pH indicator will turn red due to acidic byproducts of lactose fermentation
iii Growth of gram-positive bacteria (e.g.: Staphylococcus aureus) is inhibited by the crystal violet
dye and bile salts in the media
1 Gram-negative Enterobacteria Escherichia coli and Enterobacter aerogenes ferment lactose
2 E. coli produces pink to red colonies often with a reddish zone of bile precipitate surrounding
colonies on MacConkey's agar
3 E. aerogenes produces pink to red mucoid colonies on MacConkey's agar
4 Gram-negative bacteria Proteus vulgaris and Salmonella typhimurium grow on MacConkey's
agar, but do not ferment lactose (media appears yellow to light pink in color & colonies are
colorless; swarming of Proteus is inhibited)S
e Mannitol Salt Agar
i Partial or complete inhibition of bacterial organisms other than staphylococci
ii A change in the phenol red indicator (to yellow) means mannitol fermentation
1 Aids in the differentiation of staph species
iii S. aureus ferments mannitol and changes the color of the medium from pink to
yellow due to acid by-products of mannitol fermentation
iv Staphylococcus epidermidis grows on MSA, but does not ferment mannitol
(media remains light pink in color & colonies are colorless
v Coagulase-positive staphylococci produce growth of yellow colonies with yellow
zones. Coagulase negative staphylococci produce small red colonies with no color
vi Certain isolates of Bacillus spp. Also grow on MSA
2 Oxygen requirement tests
a Fluid thioglycollate broth
i Obligate aerobic (oxygen-needing) bacteria gather at the top of the test tube in order to absorb
maximal amount of oxygen.
ii Obligate anaerobic bacteria gather at the bottom to avoid oxygen.
iii Facultative bacteria gather mostly at the top, since aerobic respiration is
the most beneficial one; but, as lack of oxygen does not hurt them, they
can be found all along the test tube.
iv Microaerophiles gather at the upper part of the test tube but not at the top.
They require oxygen but at a low concentration.
v Aerotolerant bacteria are not affected at all by oxygen, and they are evenly
spread along the test tube.
b Anaerobic Chamber
3 Biochemical Tests a Phenol Red Broth
i Inoculate only your unknown.
ii With a sterile loop transfer some of your unknown culture and inoculate the broth aseptically
iii Fermentation will produce gas that will be trapped in the inverted durham tube and cause the
phenol red to turn yellow
1 No yellow color should occur in the control tube
2 Pseudomonas aeruginosa, Micrococcus luteus – gluc negative (red)
3 Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus – glc positive (yellow, no gas)
4 Escherichia coli , Salmonella typhimurium, Proteus mirabilis, Klebsiella pneumonia - glc
positive (yellow, with gas)
iv Homofermentative = no gas production
v Heterofermentative = gas production
b Citrate agar slants
i Inoculate only unknown
ii Transfer a small amount of your culture with a loop and make a straight
line onto the agar surface
iii Observation of a change from green to blue color indicates positive
iv P. aeruginosa- Positive
v E. coli - negative
c DNase Test
i Inoculate unknown only
ii Transfer small amount of culture with loop and make a straight line in the
iii change from green to clear color indicates positive results, the
breakdown of DNA.
iv Many isolates of S. aureus are DNAse positive.
d SIM medium agar deeps
i Inoculate only your unknown
ii Use the needle to inoculate the agar deep by transferring some of your unknown culture and
stab straight into the middle of the deep.
iii Following incubation:
a Observe for H S2production (blackening along the stab line) and motility (diffuse growth outward
from the stab line or turbidity throughout the medium). b To detect indole production, add three or four drops of Kovacs’ reagent and observe for a red
color (positive reaction).
i Enterococcus faecalis, Klebsiella pneumonia, Lactococcus lactis, Micrococcus luteus,
Staphylococcus aureus - Non-motile
ii Proteus mirabilis, Salmonella typhimurium - Motile/H2S
iii Escherichia coli, Pseudomonas aeruginosa Bacillus cereus, Enterobacter aerogenes – Motile
iv K. pneumonia
+ control for
a Catalase Test
i Obtain a pure
culture of the
ii Using pipette tip or applicator stick, pick a well-isolated colony and transfer to a glass slide.
iii Add 1 or 2 drops of the Catalase Reagent (H O )2to2the smear.
iv Examine immediately for the rapid production of gas bubbles.
v + control: E. coli
vi - control: Enterococcus faecalis
vii RESULTS: gas (O ) b2bbles indicate presence of catalase
b Oxidase Test
i Hold reagent dropper upright and POINT TIP AWAY FROM YOURSELF. Grasp the middle with
thumb and forefinger and squeeze gently to crush ampule inside the dropper.
1 Caution: Break ampule close to its center one time only. Do not manipulate dropper any further,
as the plastic may puncture and injury may occur.
ii Colonies to be tested must be isolated from other colonies.
iii The use of fresh isolates (18 – 24 h cultures) is recommended for routine testing.
iv If refrigerated, cultures must be allowed to reach room temperature prior to testing.
v One dropper is good for at least 25 reactions
1 Dispense 1 to 2 drops of Oxidase Reagent onto the tip of a cotton swab.
2 Touch the tip of the saturated swab to the top of a colony, from an actively growing culture, on
the surface of the agar medium.
3 Observe the filter paper for the development of a pink/purple color within 30 seconds.
vi Positive reactions turn the bacteria violet to purple immediately, or up to 30 s. Negative
reactions remain colorless or turn light pink / light purple after 30 s. Delayed reactions should be
vii + control: P. aeruginosa
viii - control: E. coli
c Indole Test W/ Kovac’s Reagent
Filter Paper Method: i Dispense 1 to 2 drops of Spot Indole Reagent onto a piece of filter paper
ii Using an inoculating loop, smear the growth from an actively growing culture onto the reagent-
saturated area of the filter paper.