Listeria sp. Identification Test

Listeria sp.

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Morphology: Rods

Gram: Positive

Catalase: Positive

Respiratory system: Facultative anaerobe 

Motility: Peritrichous

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Tests to identify the genus

• Hemolysis test - Sheep blood agar
• CAMP factor - Sheep blood agar
• Mixed acid or butane-diol fermentation - Clark and Lubs medium
• Sugar fermentation: xylose, mannitol, rhamnose
• Nitrate reductase

Culture

PALCAM agar

Listeria sp. on PALCAM agar

On BD PALCAM Listeria Agar, colonies of Listeria appear gray-green with a black halo. Confirmation of the presence of Listeria is made following subculture onto appropriate media and biochemical/serological identification. Colonies of mannitol-fermenting organisms such as staphylococci, which may grow on this medium, appear yellow with a yellow halo.

Refererence: BD™ [Link]

ALOA™ agar

Listeria monocytogenes on ALOA™

Please, feel free to comment if there is any mistake.

Bacillus sp. Identification Test

Bacillus sp.

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Morphologie: Rods

Gram: Positive

Catalase: Positive

Respiratory system: Facultative aerobe (B. subtilis: Obligate aerobe)

Motility: Peritrichous
Characteristic: Produces endospores

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Tests to identify spieces

• CO₂ production - Gibson Abdel Malek (GAM) medium
• Acetyl-methyl-carbinol (AMC) production - AMC medium
• Indole production - Peptone water indole-free
• Urease test - Christensen medium
• Citrate utilization - Simmons medium
• Growth at 65° C
• 7 % NaCl resistance
• Proteolysis test
• Gelatinase test
• Amylase test
• Hemolysis - Sheep blood agar
• Sugar fermentations: glucose, arabinose, mannitol and xylose

Please, feel free to comment if there is any mistake.

Clostridium sp. Identification Test

Clostridium sp.

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Morphology: Rods

Gram: Positive

Catalase: Negative
Oxidase: Negative

Respiratory system: Obligate anaerobe

Motility: Motile

Characteristic: Produces endospores

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Tests to identify the genus

• API 20A
• Rosenow medium
• Nitrate reductase test
• Proteolysis and acidification
• Sulfites reduction
• Lecithinase test

Test to identify butyric Clostridium

• Lactate utilization: butyric acid and CO₂ production - Bryant and Burkey medium

Please, feel free to comment if there is any mistake

Lactic Acid Bacteria (rods) Identification Test

Lactic acid bacteria (rods)

Lactobacillus casei, L. plantarum, L. fermentum, L. acidophilus, L. bulgaricus and L. helveticus

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Morphology: Rods

Gram: Positive

Catalase: Negative
Motility: Non-motile
Respiratory system: Facultative aerobe

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Tests to identify the genus Lactobacillus 

• CO₂ production from glucose
• Culture at 15° C and 45° C - MRS medium
• Arginine dihydrolase - Moeller medium
• Sugar fermentation : ribose, lacotse, maltose, sorbitol or sucrose

Test to identify the species

• Esculin hydrolysis - Esculin agar
• Acidification and reduction

Please, feel free to comment if there is any mistake.

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

Please, feel free to comment if there is any mistake.

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]

Please, feel free to comment if there is any mistake.

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

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Eterobacteriaceae Identification Test

Enterobacteriaceae

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Morphology: Rod-shape
Gram: Negative
Oxidase: Negative
Motility: Peritrichous (except Klebsiella sp. & Shigella sp.)
Respiratory system: Facultative anaerobe

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Tests to identify genus & species

Carbon metabolism

• Methyl red & Voges Proskauer - Clark & Lubs medium
• Glucose and lactose fermentation, gas and H₂S production - Kligler medium
• β-galactosidase - ONPG
• Citrate utilization - Simmons medium
• Malonate utilization - Leifson medium

Nitrogen metabolism

• Gelatinase test
• Amino-acids deamination
• Lysine decarboxylase - Taylor or Moeller media with and without Lysine
• Indole production - Peptone water indole-free
• Urease - Christensen medium

IMViC - Indole Methyl red Voges Proskauer Citrate

Micro-organism	Indole	MR	VP	Citrate
E. coli	+	+	-	-
Edwardsiella	+	+	-	-
Proteus sp.	+	+	-	-
Klebsiella pneumoniae	-	+	-	+
Klebsiella oxytoca	+	-	+	+
Enterobacter sp.	-	-	+	+
Serratia sp.	-	-	+	+
Citrobacter freundii	-	+	-	+
Citrobacter koseri	+	+	-	+

Please, feel free to comment if there is any mistake.