ARJUNKUMAR PJ
MSc MLT 1yr
IDENTIFICATION OF GRAM POSITIVE AND
GRAM NEGATIVE BACTERIA
Clinical sample - sample from sterile site
-???
Inoculation into culture media
Incubation overnight --??? Temp
colonies on solid media
Take a part of the colony –inoculate into
peptone water –incubate 37c – 4-6h
Uniform turbidity- Staphylococci
Surface pellicle-Pseudomonas aeruginosa
Granular turbidity- Streptococci
Solid medium:
Gram staining
Gram positive & Gram negative bacteria
Gram positive cocci
---gram positive
bacilli
Gram negative cocci ---gram negative
bacilli
Gram positive bacilli- Clostridium
Corynebacterium
Bacillus spp
Gram positive cocci
Staphylococci
Streptococci
Pneumococci
Staphylococci- pigmentation
Catalase
Mannitol fermentation
Coagulase test
Streptococci
Hemolysis on blood agar
VIRIDANS STREPTOCOCCI
STREPTOCOCCUS PYOGENES
ENTEROCOCCI
Pneumococci
Capsular swelling
Bile solubility test
Inulin fermentation
Optochin sensitivity testing
Gram negative cocci- bean shaped
intracellular
Chocolate agar in candle jar-oxidase
positive
Gonococci, meningococci
Summary Figure (Identification Scheme)
Note: S. viridans is Is alpha
hemolytic and negative for
all the tests below
below
GRAM POSITIVE COCCI
Catalase
-
+
Staphylococcus(Clusters)
Streptococcus(pairs & chains)
Coagulase
+
S. aureus

hemolytic
mannitol
yellow
-
S. epidermidis
nonhemolytic (usually)
mannitol
white
Hemolysis
BETA: Bacitracin
S.pyogenes (group A)
+
CAMP/ Hippurate
+
S. agalactiae (group B)
ALPHA: Optochin /Bile Solubility
+
S. pneumoniae
GAMMA: Bile Esculin
+
6.5% NaCl
Enterococcus
+
Bile Esculin
+
6.5% NaCl
Group D*
Non-Enterococcus
Group D*
-
(*can also be alpha hemolytic)
Gram negative bacilli
Family Enterobacteriaceae
LF
Escherichia coli, klebsiella
NLF
Salmonella, Shigella, Proteus
Oxidase , catalase, H2S, Urease, sugar
fermentation test
IMViC reaction
Testing for indole production is important in the
identification of Enterobacteria. Most strains of E. coli,
P. vulgaris, Povidencia species break down the amino
acid tryptophan with release of indole.
Principle:
The test organism is cultured in a medium which
contains tryptophan. Indole production is detected by
Kovac’s or Ehrlich reagent which contains 4(p)dimethylaminobenzaldehyde. This reacts with the
indole to produce a red colored compound.
Media used: Peptone water or Tryptone water.
Method:
The media is inoculated with the test organism, indole
paper strip is placed in the neck of the tube.
Incubate the tube at 37°C overnight.
Examine for indole production by looking for color
change in the filter paper. Yellow to pink
Indole production can also be done by adding Kovac’s
reagent to the culture and observing for the
appearance of a red color of the surface layer within 10
mins.
Controls:
Positive indole control: E. coli.
Negative indole control: Klebsiella pneumoniae,
Enterobacter aerogenes.
Controls:
Positive indole control: E. coli.
Negative indole control: Klebsiella pneumoniae, Enterobacter aerogenes.
Principle: this test detects the production of sufficient
acid during fermentation of glucose by bacteria and
sustained maintenance of a pH below 4.5.
Procedure:
the test organism is inoculated in glucose phosphate
broth and inoculated at 37°C for 2-5 days. Then add
five drops of 0.04% solution of methyl red, mix well
and read the results immediately.
Interpretation:
Positive: Red colour
Negative : Yellow color
These tubes show the results of a typical methyl red test.
Tube A. Methyl red negative
Tube B. Methyl red postive
Positive control: E. coli, Listeria monocytogenes
Negative control: Klebsiella, Enterobacter spp.
Principle: This test depends on the production of acetyl
methyl carbinol (acetoin) from pyruvic acid in the media.
In the presence of alkali and atmospheric oxygen, acetoin
is oxidized to diacetyl which reacts with α-naphthol to give
a red color.
Method: Test organism is inoculated in glucose- phosphate
broth and incubated at 37°C for 48 hrs. Then add 1 ml of
40% KOH and 3 ml of α-naphthol in absolute alcohol.
Interpretation:
Positive: Pink color within 2-5 minutes deepening to
crimson color in 30 minutes.
Negative: Colorless for 30 mins
These tubes show the results of a
typical Voges-Proskauer test.
Tube A. Voges-Proskauer positive
Tube B. Voges-Proskauer negative
Positive control: Klebsiella sp., Enterobacter sp.,
Staphylococcus.
Negative control: E. coli, Micrococcus.
Principle: It is the ability of an organism to utilize citrate as
the sole source of carbon for its growth., with resulting
alkalinity.
Procedure:
Solid Simmon’s citrate media is used. A bacterial colony is
picked up by a straight wire and inoculated into the media.
Incubated at 37°C for overnight.
Interpretation:
Positive: Growth with an intense blue color on the slant.
Negative: No growth with any change in color (green).
Positive control: Klebsiella sp., Salmonella sp. except
Salmonella typhi, Citrobacter sp.,
Negative control: E. coli, Salmonella typhi.
Principle: To determine the ability of an organism to
produce an enzyme urease which splits urea to ammonia.
Ammonia makes the medium alkaline and thus phenol red
indicator changes to pink/ red in color.
Method:
The test is done in Christensen’s urease medium. The test
organism is inoculated on the entire slope of medium and
incubated at 37°C. it is examined after 4 hours and after
overnight incubation.
Interpretation:
Positive – pink color
Negative- pale yellow.
Positive controls: Klebsiella
sp., Proteus sp., Helicobacter
pylori.
Negative control: E. coli,
Yersinia pestis.
Principle: To determine the presence of an enzyme
cytochrome oxidase which catalyses the oxidation of
reduced cytochrome by molecular oxygen.
Method:
Freshly prepared solution of 1 % tetra – methyl
paraphenylene diamine dihydrochloride (oxidase reagent)
is used. A filter paper strip, soaked in the oxidase reagent,
is smeared with test organism. In a positive oxidase
reaction the smeared area turns deep purple within 10
seconds.
Interpretation:
Positive: deep purple within 10 secs.
Negative: – No color change
Positive control:
Pseudomonas sp., Vibrio
sp., Alcaligenes sp.
Negative control: All
members of
Enterobacteriaceae.
Principle: certain bacteria have an enzyme catalase
which acts on hydrogen peroxide to relase oxygen.
Method: pick up a few colonies of test bacteria
with platinum loop from a nutrient agar plate and
mix it with a drop of H2O2 (10 volumes) on a
clean glass slide.
Interpretation:
Positive test: Immediate bubbling easily observed
Negative test: No bubbling
Positive control:
Staphylococcus, Micrococcus,
all members of
Enterobacteriaceae except
Shigella dysentriae.
Negative control: Shigella
dysenteriae typeI,
Streptococcus, Clostridium.
Principle:
To determine the ability of an organism to ferment a specific carbohydrate
(sugar) incorporated in a medium producing acid or acid with gas.
Method:
Test organism is inoculated in a sugar medium and inoculated at 37°C for
18-24 hrs. glucose, lactose, sucrose and mannitol are widely used sugars.
Sugar media contain 1% sugar. Indicator used is Andrade’s indicator
Interpretation:
Positive: pinkish –red color (acidic)
Negative: Yellow to colorless (alkaline)
Gas production can be seen as bubbles in Durham’s tube
Examples of fermentation bacteria:
Glucose fermenters: All members of the Enterobacteriaceae
Glucose and lactose fermenters: E. coli, Klebsiella sp.
Glucose and mannitol fermenter: Salmonella sp.
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Beta-hemolytic Streptococcus are spherical bacteria that
produce hemolysins capable of completely lysing (bursting
open) red blood cells.
When grown on sheep blood agar, colonies of betahemolytic Streptococcus are encircled by visible areas of
clearing where beta hemolysis has occurred.
Beta-hemolytic streptococci may be pathogens or nonpathogens. Group A Streptococcus (Streptococcus
pyogenes) causes "strep throat".
Strep throat may lead to delayed sequelae such as
rheumatic fever, glomerulonephritis, and/or scarlet fever.
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The bacitracin susceptibility test is used to
distinguish Group A streptococci, which cause
ninety percent of acute streptococcal infections in
humans, from other streptococci.
When grown on blood agar, Group A streptococci
are sensitive to (killed by) the antibiotic bacitracin .
A sterile disk impregnated with bacitracin is placed
on the first sector of an isolation plate before
incubation.
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A zone of inhibition (area with no growth) will be
seen around the disk after incubation if the organism
is a Group A beta-hemolytic Streptoccus.
Other beta-hemolytic streptococci are resistant to
(not killed by) bacitracin.
Their colonies will thus grow right up to the disk of
bacitracin.
Chocolate Agar plate
Group A Streptococcus and Group C Streptococcus
with bacitracin disks
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Coagulase is an enzyme that catalyzes the
formation of a fibrin clot in plasma.
The presence of coagulase can be detected by
heavily inoculating the test organism into
rabbit plasma and incubating the mixture for 4
to 24 hours.
Any degree of clotting during this time, from a
loose clot suspended in the plasma to a solid,
immovable clot is a positive result.
Staphylococcus aureus produces coagulase
Staphylococcus epidermidis does not.
1. Negative Coagulase Test
2. Positive Coagulase Test
Triple Sugar Iron Agar (TSI)
Are used to determine if bacteria can ferment
glucose and/or lactose and if they can produce
hydrogen sulfide or other gases.
(If an organism can ferment glucose, it is
"glucose positive".
If it ferments lactose, it is "lactose positive".)
In addition, TSI detects the ability to ferment
sucrose.
These characteristics help distinguish various
Enterobacteriacae, including Salmonella and
Shigella, which are intestinal pathogens.
TSI contains three sugars:
glucose, lactose and sucrose.
Lactose and sucrose occur in 10 times the
concentration of glucose (1.0% versus 0.1%).
Ferrous sulfate, phenol red (a pH indicator that is
yellow below pH 6.8 and red above it), and
nutrient agar are also present.
The tube is inoculated by stabbing into the agar butt
(bottom of the tube) with an inoculating wire and
then streaking the slant in a wavy pattern.
Results are read at 18 to 24 hours of incubation.
Reading the Results
A yellow slant on TSI indicates the organism ferments
sucrose and/or lactose.
On KIA a yellow slant indicates the organism ferments
lactose.
(Because KIA does not contain sucrose, sucrose fermentation
is not detected with KIA tests.)
Other results are the same for TSI and KIA. A yellow butt
shows that the organism fermented glucose.
Black preciptate in the butt indicates hydrogen sulfide
production.
Production of gases other than hydrogen sufide is indicated
either by cracks or bubbles in the media or the media being
pushed away from the bottom of the tube.
Understanding the Results
If an organism ferments glucose only, the entire tube turns
yellow due to the effect of the acid produced on phenol
red.
Because there is a minimal amount of glucose present in the
tube, the organism quickly exhausts it and begins
oxidizing amino acids for energy.
Ammonia is thus produced and the pH rises.
Within 24 hours the phenol red indicator reverts to its
original red color on the slant.
Because TSI/KIA media is poured as a deep slant, the butt
has limited oxygen and bacteria are unable to oxidize
amino acids there.
The butt thus remains yellow.
If an organism can ferment lactose and/or sucrose, the butt and slant will
turn yellow (as they do from glucose fermentation).
However, they remain yellow for at least 48 hours because of the high
level of acid products produced from the abundant sugar(s).
KIA resembles TSI in all respects except that KIA contains two sugars
(lactose and glucose) while TSI contain three sugars (lactose, glucose
and sucrose).
Like TSI media, KIA contains 10 times as much lactose as glucose.
Thus KIA tests for an organism's ability to ferment glucose or lactose but
not sucrose.
If the gas being produced is hydrogen sulfide (H2S), it reacts with the
ferrous sulfate and precipitates out as a black precipitate (ferric
sulfide) in the butt.
Organisms producing large amounts of hydrogen sulfide (e.g. Salmonella
and Proteus) may produce so much black precipitate that it masks the
yellow (acid) color of the butt
It is a differential indicator medium used for the
differentiation of lactose fermenters from non lactose
fermenters
It consists of 2% peptone, lactose, neutral red indicator
and sodium taurocholate.
It is used to distinguish lactose fermenting from Non –
lactose fermenting bacteria.