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Clinical Bacteriology Reviewer Page 1 of 17

Gram Staining ƒ ƒ ƒ

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 ƒ ƒ

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

Clinical Bacteriology Reviewer Page 2 of 17

Acid Fast Staining ƒ

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:

Procedure

Nocardia spp. and Cryptosporidium spp.

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

HEC B5MD2011 Laboratory Pictures Courtesy of Kevin Kempis

Clinical Bacteriology Reviewer Page 3 of 17

Bacterial Cultivation ƒ

Usually required for a definitive identification and characterization of the etiologic agent (Gold standard)

Purposes ƒ ƒ ƒ

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 ƒ ƒ

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

Thiosulfate Citrate-Bile Salts Agar (TCBS) ƒ

Selective and differential for Vibrio spp.

Principle ƒ ƒ

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

Yellow Colonies (A) Vibrio cholerae

Green Colonies (B) Vibrio parahaemolyticus

HEC B5MD2011 Laboratory Pictures Courtesy of Kevin Kempis

Clinical Bacteriology Reviewer Page 4 of 17

MacConkey Agar ƒ ƒ ƒ

Isolation and differentiation of lactose fermenting and non-lactose fermenting enteric bacilli Selective (Gram negative bacteria) Differential (Lactose fermenters and non-lactose fermenters)

Principle ƒ

ƒ ƒ

Inhibitors of Gram Positive bacteria ƒ Bile Salts ƒ Crystal violet ƒ Neutral Red Lactose Æ only carbohydrate source Neutral red ƒ Indicator ƒ Brown at pH 6.8-8.0 ƒ Pink-red at pH 7.6 Æ blue - 6-7.6 Æ green -