Lactic Acid Bacteria (cocci) Identification Test

Lactic acid bacteria

Lactococcus lactis, Lactococcus lactis subsp. lactis biovar diacetylactis, Lactococcus lactis subsp. cremoris, Streptococcus thermophilus, Pedicoccus acidilactici and Leuconostoc sp.

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Morphology: Cocci

Gram: Positive

Catalase: Negative
Respiratory system: Facultative anaerobe

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Tests to identify the genus

• Growth at 10° C and 45° C - Elliker/M17 broth
• NaCl resistance: 2, 4, 6 % NaCl
• CO2 production - MRS medium with bell

Tests to identify the species

• Acidification and reduction test
• Citratase test
• Arginine dihydrolase test - Moeller medium
• Sugar fermentations: glucose, sucrose, ribose, xylose, maltose, lactose

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Fecal contamination indicators

Fecal contamination indicators

Enterococcus fæcalis (Streptococcus fæcalis), Enterococcus fæcium (Streptococcus fæcium), Enterococcus durans (Streptococcus durans) and Streptococcus equinus (Streptococcus bovis)

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Morphology: Cocci

Gram: Positive

Catalase: Negative

Respiratory system: Facultative anaerobe
Characteristic: bile-resistant

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Characteristics

• Growth at 10° C and 45° C
• Temperature resistance - 60° C for 30 minutes
• Methylene blue reduction - Sherman's milk
• NaCl-resistant: 6,5 %

Identification tests

• Tellurite resistance - Potassium tellurite agar
• Tetrazolium chloride (TTC) reduction - Barnes medium
• Hemolysis - Sheep blood agar (5 %)
• Gelatinase - gelatine broth
• Sugar fermentation (lactose, sucrose, mannitol, arabinose, sorbitol)

Culture

Slanetz & Bartley medium

Selective enumeration of Enterococci in water

Sodium azide inhibits the growth of Gram-negative bacteria. TTC is an indicator of bacterial growth. It is reduced to an insoluble formazan inside the cells. This reaction is seen by the formation of red to maroon colonies.

Retain only membranes containing fewer than 100 colonies. Red, maroon or pink colonies are 

considered as characteristic. Carry out confirmation of typical colonies using Bile Esculin Azide Agar.

Reference: Biokar Diagnostics [Link]

Bile Esculin Agar

Organisms positive for esculin hydrolysis hydrolyze the esculin to esculetin and dextrose. The esculetin reacts with the ferric citrate to form a dark brown or black complex. Oxbile is used to inhibit Gram-positive bacteria other than enterococci. Beef Extract and Enzymatic Digest of Gelatin are the carbon and nitrogen sources used for general growth requirements in Bile Esculin Agar. Agar is the solidifying agent.

Reference: Neogen [Link]

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Microccus & Staphylococcus Identification Test

Microccus & Staphylococcus

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Morphology: Cocci
Gram: Positive
Catalase: Positive

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Differences between Micrococcus and Staphylococcus

Characteristics	Micrococcus spp.	Staphylococcus spp.
Respiratory system	Obligate aerobe	Facultative anaerobe
Oxidase	Positive	Negative
Resistance to vibriostatic agent O/129	S	R
Resistance to lysostaphin	R	S (±R)
Resistance to bacitracin	S	R
Growth in presence of furazolidone	+	-
% GC	66-75 %	30-39 %

Tests to identify pathogenic Staphylococci

• Free coagulase test - Coagulase broth
• Clumping factor - Slidex staph kit
• Deoxyribonuclease (DNAse) test
• Thermostable DNAse test - Lachica medium
• Hemolysis test - Sheep blood agar (5 %) 

Cultures

Baird-Parker Agar

Principles: 

Enzymatic Digest of Casein and Beef Extract are the carbon and nitrogen sources in Baird Parker Agar. Yeast Extract supplies B-complex vitamins that stimulate bacterial growth. Glycine and Sodium Pyruvate stimulate growth of staphylococci. The selectivity of the medium is due to Lithium Chloride and a 1% Potassium Tellurite Solution, suppressing growth of organisms other than staphylococci. The differentiation of coagulase-positive staphylococci is based on Potassium Tellurite and Egg Yolk Emulsion. Staphylococci that contain lecithinase break down the Egg Yolk and cause clear zones around the colonies. An opaque zone of precipitation may form due to lipase activity. Reduction of Potassium Tellurite is a characteristic of coagulasepositive staphylococci, and causes blackening of colonies. Agar is the solidifying agent.

Results:

Staphylococcus aureus on Baird-Parker agar

Coagulase-positive staphylococci produce black, shiny, convex colonies with entire margins and clear zones. Sometimes, an opaque zone of precipitation may form due to lipase activity around that clear zone. Coagulase-negative staphylococci produce poor or no growth. If growth occurs, colonies are black; clear or opaque zones are rare. The majority of other organisms are inhibited or grow poorly. If growth appears, colonies are light to brown-black, with no clear or opaque zones. 

Reference: Neogen [Link]

Baird-Parker RPF (Rabbit Plasma Fibrinogen)

Champan agar

Staphylococcus aureus on Chapman agar

Please, feel free to comment if there is any mistake