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Clinical Bacteriology Reviewer
Clinical Bacteriology Reviewer
Clinical Bacteriology Reviewer
Gram Staining
Introduced by Hans Christian Gram
Used to classify bacteria on the basis of their cellular morphologies, sizes and forms.
Permits the separation of all bacteria into two large groups, bacteria that retain the primary stain (Gram
positive) and those that take the counterstain (Gram negative).
Principle
Gram positive single layer of cell membrane with a thick cell wall
Gram negative double layer of cell membrane with a thinner peptidoglycan sandwiched between the two
cell membranes (see the image below)
Gram positive cell wall takes up the crystal violet and when followed by a mordant (iodine), it forms a
crystal violet complex within the cell. The crystal violet complex is larger than the crystal violet alone which
impedes it from being removed by the following step. Since the cell wall of the Gram negative is inside the
outer membrane, it cannot form a complex with the crystal violet even when there is already a mordant. The
primary stain stays on the outer membrane which is then washed by the alcohol allowing the safranin red to
counter stain it.
Procedure
Reagents:
Primary Stain: Crystal Violet
Mordant: Gram Iodine
Decolorizer: Ethyl Alcohol
Counterstain: Safranin Red
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Principle
The lipid capsule of the acid fast bacteria contains mycolic acid (long chain fatty acid) which is responsible for
its waxy characteristic that resists the penetration of an aqueous based solution (i.e. Crystal violet). The lipid
capsule takes up the carbolfuchsin and resists decolorization with an acid alcohol rinse. The acid fast bacteria
Other organisms that are acid-fast: Nocardia spp. and Cryptosporidium spp.
Procedure Interpretation
Number of AFB seen Report
(1000X magnification)
0 No AFB seen
1-2/300 fields Doubtful
1-9/100 fields 1+
1-9/10 fields 2+
1-9/ field 3+
>9/ field 4+
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Bacterial Cultivation
Usually required for a definitive identification and characterization of the etiologic agent (Gold
standard)
Purposes
To grow and isolate all bacteria present in a specimen
To determine which is the probable causative agent for the disease
To allow identification and characterization (therapy)
Principle
It is a process wherein the bacteria is taken from the infection site and planted into a medium that has its
nutritional and environmental requirements.
Cultivation somehow mimics the common niche of the organism.
(E.g. Vibrio spp. – Halotolerant; TCBS – contains high concentration of salt)
Principle
Inhibitors of Gram Positive and negative bacteria
Bile salts
Citrate Æ Carbohydrate source
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MacConkey Agar
Isolation and differentiation of lactose fermenting and non-lactose fermenting enteric bacilli
Selective (Gram negative bacteria)
Differential (Lactose fermenters and non-lactose fermenters)
Principle
Inhibitors of Gram Positive bacteria
Rapid Lactose Slow Lactose Non- Lactose
Bile Salts
Fermenters Fermenters Fermenters
Crystal violet
(Pink) (Pink – 48h) (Colorless)
Neutral Red
Lactose Æ only carbohydrate source Enterobacter spp. Serratia spp. Shigella spp.
Neutral red Escherichia spp. Citrobacter Salmonella spp.
Indicator Klebsiella spp. spp. Proteus spp.
Brown at pH 6.8-8.0
Pink-red at pH <6.8
Lactose Fermenters
Enterobacter aerogenes Klebsiella pneumoniae (mucoid colonies)
Eschericia coli
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Citrobacter spp.
Salmonella spp.
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Lactose Fermenters
Escherichia coli (green metallic sheen) Klebsiella pneumoniae
Non-Lactose Fermenters
Proteus spp.
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Principle
Inhibitors of Gram Positive bacteria Red colonies Colorless w/o Colorless w/
Bile Salts black centers black centers
Inhibitors of other Gram Negative bacteria
Brilliant green
Bile Salts Coliforms Shigella spp. Salmonella spp.
Lactose Æ only carbohydrate source
Sodium thiosulfate Æ Sulfur source (H2S
production)
Ferric sulfide Æ black color
** H2S + Ferric Ammonium citrate = Ferric sulfide
Shigella spp.
Salmonella spp.
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Principle
Fresh sterile sheep’s blood is added to the medium after autoclaving and just before the medium solidify
** Chocolate agar is made by adding the blood right after the autoclaving when the medium is still hot
Presence of blood determines the hemolytic capacity of an organism
Hemolysis is differentiated into Alpha(partial), Beta(Complete) and Gamma(None)
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Principle
Inhibitors of Gram Positive bacteria
Sodium deoxycholate
- Partially inhibits E .coli
- Inhibits Proteus swarming
Xylose, Lactose and Sucrose (Carbohydrate source)
- Fermented by all the members of Enterobactericeae except for Shigella spp.
Phenol red Æ indicator
- Fermentation of Carbohydrate Æ yellow
Lysine
- Lysine positive turns yellow to red via decarboxylation
H2S Positive
- Reaction of H2S with ferric ammonium citrate Æ black centered colonies
Interpretation
Reaction Microorganisms
Red Colonies Shigella spp.
Red Colonies with black centers (H2S +) Salmonella spp
Yellow Colonies Escherichia coli
Yellow Colonies with black centers (H2S +) Citrobacter spp. and Proteus spp.
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Principle
Inhibitors of Gram positive and negative bacteria
- Bile salts
Carbohydrate source Lactose Non-lactose H2S
- Lactose fermenting fermenters Producers
- Sucrose Escherichia coli Shigella spp. Salmonella spp.
- Salicin Yersinia spp.
Bromthymol blue Æ indicator
- >7.6 Æ blue
- 6-7.6 Æ green
- <6 Æ yellow (fermentation of any sugar)
Sodium thiosulfate
- H2S production Æ black centered colonies
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**gas production is
observed by the presence of
bubbles or cracks
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Principle
Indicator Æ bromcresol purple
Glucose Æ Turns to yellow when fermented (seen in the butt)
Ability to produce H2S gas Æ
Deamination (slant) **A before C: deAmination before deCarboxylation : Slant before Butt
- Occurs in the presence of oxygen (Aerobic process)
- Ammonia produced will react with ferric ammonium citrate Æ dark red on slant
- Negative deamintaion Æ remain purple
Decarboxylation (butt)
- Occurs in the absence of Oxygen (anerobic process)
- Produces an alkaline environment ( yellow to purple: Æ )
A – K/K
B – K/K H2S +
C – K/A
D – R/A
E – Uninoculated
Slant (deamination)
(+) dark Red
(-) Purple
Butt (decarboxylation)
(+) Purple
(-) Yellow
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Principle
Inhibitors of Gram Positive bacteria
Eosin
Methylene blue
Sodium Citrate Æ only carbohydrate source
- Sodium citrate Æ ammonia Æ ammonium hydroxide (alkaline) Æ Prussian blue
Prussian blue color reaction is observed with the top test tube inoculated with K. pneumoniae.
The bottom test tube is uninoculated (normal color of green)
Indole Broth
Determine the ability of an organism to split tryptophan to form the compound indole
Principle
Tryptophan present in peptone is oxidized into indole by Tryptophanase
Indole + p-dimethylaminobenzaldehyde (Kovac’s or Erlich’s) Æ red color between the rgt and broth
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Principle
Bromcresol purple Æ indicator
Agar used is semisolid (allows more movement of the bacteria)
Contains tryptophan for production
Contains Ornithine for detection of its decarboxylation
Interpretation
Test Reaction Organisms
Motility (+) diffused growth Proteus spp.
Salmonella spp.
(-) growth along the line Klebsiella spp.
Shigella spp.
Indole production (+) red ring Escherichia coli
(-) absence of red ring Klebsiella spp.
Ornithine decarboxylation (+) Purple ?
(-) Yellow ?
MIO medium
(+) Motility
(+) Indole
(+) Motility
(+) Motility and
(-) Ornithine decarboxylation
(-) Motility
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Principle
Urease catalyzes the hydrolysis of urea to produce ammonia and CO2
Phenol red Æ indicator
(+) for ammonia production reacts in the solution to produce ammonium carbonate that shifts the pH
from 6.8 (light orange or Salmon pink?) to 8.1 (magenta or pink-red or Fuchsia pink)
Positive
reaction
Oxidase Test
Determine the presence of bacterial cytochrome oxidase
Principle
Oxidation of tetramethyl-p-phenylenediamine dihydrochloride Æ indophenol (dark purple)
Production of intense dark purple color after the addition of the substrate Æ positive for oxidase
** Wires used for transferring organism to slide yield false positive result. So in the laboratory, a wooden
applicator stick is used to transfer the organism from the tube or plate to the slide.
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Principle
Glucose is fermented to pyruvic acid by one of the two pathways Æ (+)MR or (+)VP
First pathway (detected by MR)
o Lactic acid, acetic acid, formic and succinic acid Æ decrease in pH Æ production of red color
Second pathway (detected by VP)
o Acetylmethyl carbinol (Acetoin)
Neutral end product
Acetoin + O2 + 40% KOH Æ Diacetyl Form
Diacetyl form + alpha napthol (added reagent) Æ production of red color
Must be incubated for 48 hrs
Methyl Red Voges-Proskauer
Organism MR VP
Escherichia coli + -
Enterobacter spp. - +
Principle
Beta galactosidase hydrolyzes ONPG Æ orthonitrophenol (yellow color)
Positive Negative
Escherichia coli Salmonella spp.
Positive
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Notes:
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