Bacterial Culture Media In Plate & Tube

BACTERIOLOGY 231 PLATE MEDIA MEDIA

CARBOHYDRATE

INHIBITOR

pH INDICATOR

BAP Blood agar

Alpha hemolysis

RESULT Incomplete or partial lysis with greening S. pneumoniae

Outdated human blood or 5% defribinated sheep blood (preferred) Trypticase Soy Agar or Nutrient agar

Beta hemolysis { Nonselective agar for Hemolysis Differentiation }

S. pyogenes S. agalactiae

PINHEAD – Staphylococcus PINPOINT - Streptococcus

Gamma hemolysis

CAP Chocolate agar

Complete lysis with colorless zone [diffusion of hemoglobin into the surrounding medium]

Nonhemolytic

Hemolytic Lysed red blood cells Trypticase Soy Agar or Nutrient agar X factor (hemin) V factor or NAD (nicotinamide adenine dinucletide, coenzyme)

{Nonselective agar for Hemolysis Differentiation} PINHEAD – Staphylococcus PINPOINT – Streptococcus **S. aureus - 1° src. of X factor

Nonhemolytic

PEA Phenylethyl alcohol agar

Bacillus anthracis (24° incubation) **Most Bacillus sp. grow

{Selective & Differential agar} Gram (-) inhibitor Inhibits swarming of Proteus

✔ Yellow colonies [pH 6.8]

MSA Mannitol salt agar

Coagulase (+) S. aureus Some Micrococcus & Enterococcus

{Selective & Differential agar} Inhibits Gram (-)

Mannitol

7.5% NaCl

Phenol red

✖Small red colonies with red/purple zone [pH 8.4]

Coagulase (-) Staph. sp. (episodemidis/saprophiticus) Most Gram (-)

✔Pink to purple colonies with Green metallic sheen

E. coli (Rapid Lactose Fermenter - RLF)

Beef extract

EMB Eosin methylene blue agar {Selective & Differential agar} Inhibits Gram (+)

Lactose

Eosin Methylene blue

✔Pink to purple colonies with Green metallic sheen w/ or w/o black center (fisheye) ✔ Pink to purple colonies, mucoid

Enterobacter aerogenes (RLF) Motile*

Klebsiella pneumonia (RLF) Nonmotile*

✖Yellow to colorless (pink) colonies (NLF)

MAC MacConkey agar

✔Pink to red colonies [pH <6.8]

{Differential agar} Inhibits Gram Lactose

Crystal violet Bile salts

Neutral red

✖Yellow to colorless colonies [pH 6.8-8.0]

(+) HEA Hektoen Enteric agar {Selective & Differential agar} Inhibits normal flora coliform of the lower GI tract [Salmonella and Shigella species from other Gram (-) enteric organisms]

Lactose Sucrose Salicin

Bile salts Citrate

Bromthymol blue [H2S: ferric ammonium chitrate; sodium thiosulfate]

✔Yellow to orange w/ black center ✔Yellow to orange w/o black center

✖Blue to green w/o black center ✖Blue to green w/ black center

Salmonella typhi Shigella sonnei Proteus vulgaris Psedomonas aeroginosa RLF: Escherichia, Enterobacter, Klebsiella LLF: Citrobacter, Serratia, Hafnia alvei, Y. enterocolitica, S. arizonae, S. sonnei NLF: Proteus, Providencia, Morganella, Edwardsiella Salmonella (r/o) arizonae Shigella (r/o) sonnei Yersenia (r/o) enterocolitica Citrobacter freundii Salmonella arizonae E. coli Klebsiella pneumonia Enterobacter aerogenes Shigella sonnei Serratia marcesens Providencia rettegri Morganella morganii Shigella dysenteriae Salmonella typhi Proteus vulgaris Proteus mirabilis

XLD Xylose-lysine deoxycholate

Phenol red [H2S: ferric ammonium chitrate; sodium thiosulfate]

{Selective & Differential agar} Inhibits normal flora coliforms Xylose [Partially E. coli & swarming of Proteus]

SSA SalmonellaShigella agar

{Selective & Differential agar} Inhibition of normal flora coliforms Inhibits Gram (+) & (-)

Lactose

Na+ Deoxycholate

Bile salts Brilliant green

[Lysine AA → Lysine decarboxylase ] (+)LDC = CADAVERIN (pH)

Neutral red [H2S: ferric ammonium chitrate; sodium thiosulfate]

✔ Yellow colonies w/ black center (acid) ✔Yellow colonies w/o black center (acid)

✖Red colonies w/ black center (alk) ✖Red colonies w/o black center (alk) ✔Pink to red colonies (LF)

✖Yellow to colorless w/o black center ✖Yellow to colorless w/ black center ✔Yellow colonies

TCBS Thiosulfate Citrate Bile salt Sucrose agar

✖Blue-green colonies

{Selective & Differential agar} Isolate Vibrio sp.

Sucrose

Sodium citrate Sodium thiosulfate Oxgall

Bromthymol blue [H2S: ferric ammonium chitrate; sodium thiosulfate]

Citrobacter freundii Proteus vulgaris Proteus mirabilis E. coli Klebsiella pneumonia Enterobacter aerogenes Providencia retteri Serratia marcesens

Salmonella typhi Shigella dysenteriae Normal flora coliforms *Shigella sonnei *Salmonella arizonae (w/ black center) Shigella dysenteriae Shigella flexneri Shigella boydii Salmonella typhi V. cholera V. alginolyticus V. parahaemolyticus V. vulnificus Aeromonas Psuedomonas

BSA Bismuth sulfate agar

{Selective & Differential agar} Isolate Salmonella sp. Inhibit Gram (+), lactose fermenter & shigellase

Bismuth sulfate Brilliant green (Inhibts other enteric)

[H2S: ferric sulfide] soluble

✖ Black colonies with metallic sheen (w/ ring)

Salmonella typhi

✔Yellow to orange colonies

S. gallinarum S. choleraesuis S. paratyphi

CYSTEINE TELLURITE AGAR (CTA) >gray to black colonies Corynebacterium diptheriae

Francisella tularensis Legionella pneumophilia Clostridium difficile Prevotella melaninogenica 5-6 wks: LEPTOSPIRA 3-4 wks: BRUCELLA 2-3 wks: MYCOBACTERIUM BACTERIOLOGY 231

MODIFIED TYNSDALE AGAR >black colonies with brown halos LOEFFLER SERUM SLANT >enhance METACHROMATIC granules BLOOD CYSTEIN GLUCOSE AGAR BUFFERED CHARCOAL YEASE EXTRACT (BYCE) With L-cysteine CYCLOSERINE CEFOXITIN FRUCTOSE AGAR (CCFA) (Brucella) LAKED KANAMYCIN VANCOMYCIN 5% SHEEP BLOOD AGAR (LKV) BIPHASIC (2 phased media) >SEPTI-CHECK & OLD CASTAÑEDA *ideal for airborne microbes

TUBE MEDIA MEDIA

TSI Triple Sugar Iron agar

FORMATION

Butt Slant

CARBOHYDRATE

Glucose [Butt] Lactose [Slant] Sucrose [Slant]

pH INDICATOR

Phenol red

Decarboxylation (slant) = Alkaline (red)

H2S INDICATOR

RESULT A/AG

Ferrous ammonium sulfate

A/AG+ or K/AG+

A/AG or K/AG

K/AG+

A/A or K/A K/A

K/A+ K/K

Legend: A(yellow); K(red); G(gas production); +(H2S production)

E. coli Enterobacter aerogenes Pantoea agglomerans Klebsiella pneumonia Serratia Salmonella arizonae Citobacter freundii Salmonella arizonae Hafnia alvei Morganella morganii Morganella freundii Citrobacter koseri Serratia marcescens Hafnia alvei Proteus vulgaris Proteus mirabilis Salmonella paratyphi Salmonella choleraesuis Edwardsiella tarda Yersinia entericolitica Shigella sonnei Shigella dysenteriae Shigella flexneri Shigella boydii Salmonella typhi Pseudonmonas aeroginosa Alcaligenes

MEDIA LIA Lysine Iron Agar

FORMATION

Butt Slant

AMINO ACID

Lysine

CARBOHYDRATE

Glucose

pH INDICATOR

H2S INDICATOR

Bromcresol purple

Ferric ammonium citrate

RESULT

K/K

K/A

R/A *deaminase(+)

K/AG+

Legend: K(purple); A(yellow); R(red); +(H2S production)

E. coli Enterobacter aerogenes Klebsiella pneumonia Serratia marcescens Hafnia alvei Pantoea agglomerans Citobacter freundii Shigella dysenteriae Shigella flexneri Shigella boydii Proteus Providencia Morganella Salmonella typhimurium Salmonella arizonae Salmonella typhi Salmonella parayphi Salmonella choleraesuis Edwardsiella tarda

MEDIA

Indol Production Test

Methyl Red Test MIXED ACID PATHWAY

Voges Proskauer Test BUTYLENE GLYCOL PATHWAY

PRINCIPLE The organism oxidized tryptophan by trytophanase ↓ tryptophan ↓ pyruvic acid ↓ amino acid ↓ indol

The organism ferments glucose to pyruvic acid resulting in mixed acid products leading to decreased pH (4.55.5) of the medium and takes the color of the indicator

The organism ferments glucose producing acetylmethylcarbinol (acetoin) and converted to dimethyl/diacetyl in the presence of oxygen and KOH. KOH → reacts w/ GUANIDINE = color

FORMATION

MEDIUM

Butt

Tryptone broth

Butt

MRVP (Methyl Red Voges Proskauer) medium

Butt

MRVP

REAGENT ①Ehrlich’s Add: EtOH and/or XYLENE (xtract indol) then p-dab ②Kovac’s p-aminodimethyl benzaldehyde (p-dab) ③Spot Indole: rapid detection – filter paper:1% pdab in 4 hours.

Methyl red indicator

VP Reagent A: Alpha napthol in absolute methyl alcohol VP Reagent B: 40% KOH with 0.3% creatine

POSITIVE

NEGATIVE

Red Ring

Yellow/Brown Ring

Red solution

Yellow solution

Pink to Red color

Yellow or copper-like

MEDIA

PRINCIPLE

FORMATION

MEDIUM

INDICATOR

POSITIVE

NEGATIVE

Citrate Utilization Test

The organism utilizes the citrate (sole src. of carbon) producing ammonia and converted to ammonium hydroxide. This alkaline compound raises the pH of the medium and takes the blue color.

Slant

SCA (Simmon Citrate Agar)

Bromthymol blue

Prussian blue color

Green color

Urease Test

Urease splits the urea molecules into ammonia, carbon dioxide and water. Ammonia reacts in solution to form alkaline compound, ammonium carbonate, which results in an increased pH of the medium and a color change in the indicator to pink-red.

Butt (Broth)

Urea broth

Phenol red

Pink-red

No color change

Sulfide indole motility (SIM)

Butt (Semisolid)

H2S: ferric ammonium chitrate; sodium thiosulfate

Motile (growth Nonmotile pattern – (growth away from pattern is inoculation confined) line) H2S production (Blackening of the butt) Indol production (red ring)

Water Analysis Testing >sanitary supply of water >suitability for general use Method: Multiple Fermentation Tube Technique

Brilliant Green Bile Lactose Broth

Uninocculated

PRESUMPTIVE TEST: LACTOSE BROTH CONFIRMED TEST: BGBL, EMB, ENDO AGAR COMPLETED TEST: LACTOSE BROTH

Positive E. coli + Gas Production + Turbidity