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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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Acid Fast Staining
Allows the detection of acid fast bacteria

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 (1000X magnification) 0 1-2/300 fields 1-9/100 fields 1-9/10 fields 1-9/ field >9/ field Report No AFB seen Doubtful 1+ 2+ 3+ 4+

Seen in this picture are the Acid fast bacteria (Mycobacterium tuberculosis) stained as red (arrows) with the surrounding blue background

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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)

Classification and Functions
A. Enrichment Contain specific nutrients required for the growth of a particular bacterial pathogens that may be present alone or with other bacteria Buffered charcoal-yeast extract agar (L-cysteine) – Legionella pneumophila B. Supportive Contain nutrients for the growth of different nonfastidious organisms without promoting any of the other organism’s growth Nutrient Agar C. Selective Contain agents that are inhibitory to all the organisms except those wanted microorganism TCBS – Vibrio spp. D. Differential Contain certain factor that allows different strains of bacteria to exhibit certain metabolic or culture characteristic when grown sBAP (sheep’s blood agar plate) – differentiates Streptococcus spp.

Thiosulfate Citrate-Bile Salts Agar (TCBS)
Selective and differential for Vibrio spp.

Principle
Inhibitors of Gram Positive and negative bacteria Bile salts Citrate Carbohydrate source

Yellow Colonies (A) Vibrio cholerae

Green Colonies (B) Vibrio parahaemolyticus

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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 Bile Salts Crystal violet Neutral Red Lactose only carbohydrate source Neutral red Indicator Brown at pH 6.8-8.0 Pink-red at pH <6.8 Rapid Lactose Fermenters (Pink) Enterobacter spp. Escherichia spp. Klebsiella spp. Slow Lactose Fermenters (Pink – 48h) Serratia spp. Citrobacter spp. Non- Lactose Fermenters (Colorless) Shigella spp. Salmonella spp. Proteus spp.

Lactose Fermenters
Enterobacter aerogenes

Klebsiella pneumoniae (mucoid colonies)

Eschericia coli

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Clinical Bacteriology Reviewer Page 5 of 17 Late Lactose Fermenters
(24hrs- NLF and 48hrs- LF) Citrobacter spp.

Non-Lactose Fermenters
Proteus mirabilis (swarming colonies)

Serratia marcescens (red pigment production)

Pseudomonas aeruginosa

Salmonella spp.

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Eosin Methylene Blue Agar (EMB)
Isolation and differentiation of lactose-fermenting and non-lactose fermenting enteric bacilli For Escherichia coli identification

Principle
Inhibitors of Gram Positive bacteria Eosin Methylene blue Lactose only carbohydrate source

Lactose Fermenters (Purple) Escherichia coli (Green metallic sheen) Klebsiella spp. Serratia marcescens Enterobacter aerogenes

Non-Lactose Fermenters (colorless) Proteus spp. Pseudomonas aeruginosa

Lactose Fermenters
Escherichia coli (green metallic sheen) Klebsiella pneumoniae

Non-Lactose Fermenters
Proteus spp.

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Salmonella Shigella Agar
Selective for Salmonella and Shigella spp.

Principle
Inhibitors of Gram Positive bacteria Bile Salts Inhibitors of other Gram Negative bacteria Brilliant green Bile Salts Lactose only carbohydrate source Sodium thiosulfate Sulfur source (H2S production) Ferric sulfide black color ** H2S + Ferric Ammonium citrate = Ferric sulfide Red colonies Colorless w/o black centers Shigella spp. Colorless w/ black centers Salmonella spp.

Coliforms

Shigella spp.

Salmonella spp.

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Sheep’s Blood Agar Plate (BAP)
Allows the cultivation of fastidious microorganisms Differential medium and NOT selective Determines hemolytic capacity of different organisms

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) Alpha Beta Gamma

Alpha α Streptococcus pneumoniae

Beta β Streptococcus pyogenes

Gamma γ Enterococcus faecalis

Optochin Test
S. pneumoniae(S) vs Viridian Strep (Resistant)

Bacitracin Test
S. pyogenes (S) vs. S. agalactiae (Resistant)

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Xylose Lysine Desoxycholate Agar (XLD)
Isolation and differentiation of Salmonella and Shigella spp. From other gram-negative enteric bacilli Isolation and differentiation of stool pathogens Differs from HE by the ability to detect Lysine decarboxylation

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 indicator - Fermentation of Carbohydrate yellow Lysine - Lysine positive turns yellow to red via decarboxylation H2S Positive - Reaction of H2S with ferric ammonium citrate black centered colonies

Interpretation
Reaction Red Colonies Red Colonies with black centers (H2S +) Yellow Colonies Yellow Colonies with black centers (H2S +) Microorganisms Shigella spp. Salmonella spp Escherichia coli Citrobacter spp. and Proteus spp.

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Hektoen Enteric Agar (HE)
Differential, selective medium for Shigella and Salmonella spp. From other gram-negative enteric bacilli Preferentially allows the growth of stool pathogens by inhibiting the enteric normal flora

Principle
Inhibitors of Gram positive and negative bacteria - Bile salts Lactose Carbohydrate source fermenting - Lactose Escherichia coli - Sucrose Yersinia spp. - Salicin Bromthymol blue indicator - >7.6 blue - 6-7.6 green - <6 yellow (fermentation of any sugar) Sodium thiosulfate - H2S production black centered colonies Non-lactose fermenters Shigella spp. H2S Producers Salmonella spp.

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Triple Sugar Iron Agar (TSI)
Initial step for the identification of Enterobactericeae Used to determine whether a gram-negative rod utilizes glucose and lactose or sucrose fermentatively and forms hydrogen sulfide H2S.

Principle
Fermentation of Glucose makes slant and butt yellow, but the slant reverts to alkaline (red) due to the oxidative decarboxylation. (K/A) Fermentation of Glucose + Lactose or Sucrose counteracts the alkalinity on the slant and yields a yellow color (A/A) Carbohydrate source (Triple Sugar) o Lactose o Sucrose o Glucose Ferrous sulfate indicator for H2S gas production Phenol red indicator C-- Control 1 – K/K 2 – K/A 3 – K/@ H2S + 4 – A/@ 4A – K/@ 5 – A/@ H2S + **gas production is observed by the presence of bubbles or cracks Reaction A/@ H2S + A/@ H2S K/@ H2S + K/A H2S Fermented Carbohydrate Glucose and Lactose and/or Sucrose Glucose and Lactose and/or Sucrose Glucose only Glucose only Organisms Citrobacter freundii Escherichia coli Klebsiella spp. Enterobacter spp. Salmonella spp. Proteus spp. Citrobacter spp. Shigella spp. Providencia spp. Serratia spp. Anaerogenic E. coli Pseudomonas spp.

K/K H2S -

None

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Lysine Iron Agar (LIA)
Determines the ability of the bacteria to deaminase, decarboxylase lysine and also to produce H2S Useful in identification of Salmonella, Shigella, Proteus, Providencia and Morganella spp. Proteus – only member of the Enterobactericeae Deaminase lysine

Principle
Indicator Glucose bromcresol purple Turns to yellow when fermented (seen in the butt) Ability to produce H2S gas 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 - Negative deamintaion remain purple Decarboxylation (butt) - Occurs in the absence of Oxygen (anerobic process) - Produces an alkaline environment ( yellow to purple: A – K/K B – K/K H2S + C – K/A D – R/A E – Uninoculated Slant (deamination) (+) dark Red (-) Purple Butt (decarboxylation) (+) Purple (-) Yellow Organism Escherichia coli Proteus mirabilis Pseudomonas spp E. aerogenes Shigella spp. Salmonella spp. Lysine deamination + Lysine decarboxylation + + + H2S Gas + ) dark red on slant

K/A: Negative deamination, Negative decarboxylation Shigella spp.

K/K H2S+: Negative deamination, Positive decarboxylation Salmonella spp.

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Simmon’s Citrate Agar (SCA)
Determines if the bacteria can utilize citrate as its own carbon source

Principle
Inhibitors of Gram Positive bacteria Eosin Methylene blue Sodium Citrate only carbohydrate source - Sodium citrate ammonia ammonium hydroxide (alkaline) Prussian blue

Positive reaction (Prussian Blue or with Growth) Klebsiella spp.

Negative reaction (Green color or NO growth) Escherichia coli

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 Tryptophan indole + pyruvic acid + ammonia

Reagents used in detecting indole (dropped o Erlich’s reagent (more sensitive) used in Anaerobic and nonfermentative organisms o Kovac’s reagent Indole + p-dimethylaminobenzaldehyde (Kovac’s or Erlich’s) Indole Positive Escherichia coli Proteus vulgaris Indole Negative Enterobacter spp. Klebsiella spp. Proteus mirabilis red color between the rgt and broth

Presence of red ring between the reagent and the broth = Positive

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Motility Indole Ornithine Medium (MIO)
Determines the motility of the bacteria as well as its indole production and ornithine decarboxylation

Principle
Bromcresol purple indicator Agar used is semisolid (allows more movement of the bacteria) Contains tryptophan for production Contains Ornithine for detection of its decarboxylation Interpretation Test Motility Reaction (+) diffused growth (-) growth along the line Indole production Ornithine decarboxylation MIO medium (+) red ring (-) absence of red ring (+) Purple (-) Yellow Organisms Proteus spp. Salmonella spp. Klebsiella spp. Shigella spp. Escherichia coli Klebsiella spp. ? ?

(+) Motility (+) Indole (+) Motility (+) Motility and (-) Ornithine decarboxylation (-) Motility

Motility Agar w/o Bromcresol purple 1% triphenyltetrazolium chloride (responsible for the red color)

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Christensen’s Agar or Stuart Broth (Urease Test)
Determines the ability of an organism to produce urease

Principle
Urease catalyzes the hydrolysis of urea to produce ammonia and CO2 Phenol red 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) Urease Positive (Fuchsia Pink) Rapid (w/in 4 hrs) Slow Proteus spp. Enterobacter spp. Morganella spp. Klebsiella pneumoniae Stuart Broth Urease negative (Salmon pink?) Escherichia coli

Christensen’s Agar

Positive reaction

Oxidase Test
Determine the presence of bacterial cytochrome oxidase

Principle
Oxidation of tetramethyl-p-phenylenediamine dihydrochloride indophenol (dark purple) Production of intense dark purple color after the addition of the substrate 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.

Positive (Purple) Vibrio spp. Neisseria spp.

Negative Escherichia coli

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Methyl Red / Voges-Proskauer (MRVP) Test
Determine the ability of an organism to produce and maintain stable acid and neutral end products from glucose fermentation and to overcome the buffering capacity of the system.

Principle
Glucose is fermented to pyruvic acid by one of the two pathways (+)MR or (+)VP First pathway (detected by MR) o Lactic acid, acetic acid, formic and succinic acid decrease in pH production of red color Second pathway (detected by VP) o Acetylmethyl carbinol (Acetoin) Neutral end product Acetoin + O2 + 40% KOH Diacetyl Form Diacetyl form + alpha napthol (added reagent) production of red color Must be incubated for 48 hrs

Methyl Red

Voges-Proskauer

Positive

Negative Organism Escherichia coli Enterobacter spp. MR + VP +

Positive

Negative

ONPG (o-Nitrophenyl-B-D-Galactopyranoside) Test
Determine the ability of the organism to produce beta-galactosidase

Principle
Beta galactosidase hydrolyzes ONPG orthonitrophenol (yellow color)

Positive Escherichia coli

Negative Salmonella spp.

Positive

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Notes:

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