clinical bacteriology reviewer

Clinical Bacteriology Reviewer of 17

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

HEC B5MD2011 Laboratory Pictures Courtesy of Kevin Kempis


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)

Report

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+

Seen in this picture are the Acid fast bacteria (Mycobacterium

tuberculosis) stained as red (arrows) with the

surrounding blue background



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)

Green Colonies (B)

Vibrio cholerae Vibrio parahaemolyticus



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

Lactose Fermenters Enterobacter aerogenes

Eschericia coli

Klebsiella pneumoniae (mucoid colonies)

Rapid Lactose Fermenters (Pink)

Slow Lactose Fermenters (Pink – 48h)

Non- Lactose Fermenters (Colorless)

Enterobacter spp. Escherichia spp. Klebsiella spp.

Serratia spp. Citrobacter spp.

Shigella spp. Salmonella spp. Proteus spp.



Late Lactose Fermenters (24hrs- NLF and 48hrs- LF) Citrobacter spp.

Serratia marcescens (red pigment production)

Non-Lactose Fermenters Proteus mirabilis (swarming colonies)

Pseudomonas aeruginosa

Salmonella spp.



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 Escherichia coli (green metallic sheen)

Klebsiella pneumoniae

Non-Lactose Fermenters Proteus spp.

Lactose Fermenters (Purple)

Non-Lactose Fermenters (colorless)

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

Proteus spp. Pseudomonas aeruginosa



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

Shigella spp.

Salmonella spp.

Red colonies Colorless w/o black centers

Colorless w/ black centers

Coliforms Shigella spp. Salmonella spp.



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 Beta β Gamma γ Streptococcus pneumoniae Streptococcus pyogenes Enterococcus faecalis Optochin Test S. pneumoniae(S) vs Viridian Strep (Resistant)

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



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 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.



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

Carbohydrate source - Lactose - Sucrose - Salicin

Bromthymol blue indicator - >7.6 blue - 6-7.6 green - <6 yellow (fermentation of any sugar)

Sodium thiosulfate - H2S production black centered colonies

Lactose fermenting

Non-lactose fermenters

H2S Producers

Escherichia coli Yersinia spp.

Shigella spp. Salmonella spp.



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

Reaction Fermented Carbohydrate Organisms A/@ H2S + Glucose and Lactose and/or Sucrose Citrobacter freundii A/@ H2S - Glucose and Lactose and/or Sucrose Escherichia coli

Klebsiella spp. Enterobacter spp.

K/@ H2S + Glucose only Salmonella spp. Proteus spp. Citrobacter spp.

K/A H2S - Glucose only Shigella spp. Providencia spp. Serratia spp. Anaerogenic E. coli

K/K H2S - None Pseudomonas spp.

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



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 bromcresol purple Glucose 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 dark red on slant

- Negative deamintaion remain purple Decarboxylation (butt)

- Occurs in the absence of Oxygen (anerobic process)

- Produces an alkaline environment ( yellow to purple: )

Organism Lysine deamination

Lysine decarboxylation

H2S Gas

Escherichia coli - + - Proteus mirabilis + - - Pseudomonas spp - - - E. aerogenes - + - Shigella spp. - - - Salmonella spp. - + +

A – K/K B – K/K H2S + C – K/A D – R/A E – Uninoculated Slant (deamination) (+) dark Red (-) Purple Butt (decarboxylation) (+) Purple (-) Yellow

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

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



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)

Negative reaction (Green color or NO growth)

Klebsiella spp. 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) red color between the rgt and broth

Indole Positive Indole Negative Escherichia coli Proteus vulgaris

Enterobacter spp. Klebsiella spp. Proteus mirabilis

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



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 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

(+) Motility and (-) Ornithine decarboxylation

(+) Indole

(+) Motility

(-) Motility

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



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)

Stuart Broth

Christensen’s Agar

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.

Urease Positive (Fuchsia Pink) Urease negative (Salmon pink?) Rapid (w/in 4 hrs) Proteus spp. Morganella spp.

Slow Enterobacter spp. Klebsiella pneumoniae

Escherichia coli

Positive reaction

Positive (Purple) Negative Vibrio spp. Neisseria spp.

Escherichia coli



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

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)

Organism MR VP Escherichia coli + - Enterobacter spp. - +

Positive Negative Escherichia coli Salmonella spp.

Negative Positive Positive Negative

Positive



Notes:


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