BACTERIOLOGY 231 PLATE MEDIA

MEDIA CARBOHYDRATE INHIBITOR pH INDICATOR RESULT

BAP Blood agar

Outdated human blood or 5% defribinated sheep blood

(preferred)

Trypticase Soy Agar or Nutrient agar

{ Nonselective agar for Hemolysis Differentiation }

PINHEAD – Staphylococcus PINPOINT - Streptococcus

Alpha hemolysis

Incomplete or partial lysis with greening S. pneumoniae

Beta hemolysis

Complete lysis with colorless zone [diffusion of hemoglobin into the surrounding medium] S. pyogenes S. agalactiae

Gamma hemolysis

Nonhemolytic

CAP Chocolate agar

Lysed red blood cells

{Nonselective agar for Hemolysis Differentiation}

PINHEAD – Staphylococcus PINPOINT – Streptococcus

**S. aureus - 1° src. of X factor

Hemolytic

Trypticase Soy Agar or Nutrient agar

X factor (hemin)

V factor or NAD (nicotinamide adenine dinucletide,

coenzyme)

Nonhemolytic

PEA Phenylethyl alcohol agar

{Selective & Differential agar} Gram (-) inhibitor

Inhibits swarming of Proteus

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

MSA Mannitol salt agar

{Selective & Differential agar} Inhibits Gram (-)

Mannitol 7.5% NaCl Phenol red

✔ Yellow colonies [pH 6.8]

Coagulase (+) S. aureus Some Micrococcus & Enterococcus

Beef extract

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

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

EMB Eosin methylene blue agar

{Selective & Differential agar} Inhibits Gram (+)

Lactose

Eosin Methylene blue

✔Pink to purple colonies with Green metallic sheen

E. coli (Rapid Lactose Fermenter - RLF)

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

Enterobacter aerogenes (RLF) Motile*

✔ Pink to purple colonies, mucoid

Klebsiella pneumonia (RLF) Nonmotile*

✖Yellow to colorless (pink) colonies (NLF)

Salmonella typhi Shigella sonnei Proteus vulgaris Psedomonas aeroginosa

MAC MacConkey agar

{Differential agar} Inhibits Gram

(+)

Lactose Crystal violet

Bile salts Neutral red

✔Pink to red colonies [pH <6.8]

RLF: Escherichia, Enterobacter, Klebsiella LLF: Citrobacter, Serratia, Hafnia alvei, Y. enterocolitica, S. arizonae, S. sonnei

✖Yellow to colorless colonies [pH 6.8-8.0]

NLF: Proteus, Providencia, Morganella, Edwardsiella Salmonella (r/o) arizonae Shigella (r/o) sonnei Yersenia (r/o) enterocolitica

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

Citrobacter freundii Salmonella arizonae

✔Yellow to orange w/o black center

E. coli Klebsiella pneumonia Enterobacter aerogenes Shigella sonnei Serratia marcesens

✖Blue to green w/o black center

Providencia rettegri Morganella morganii Shigella dysenteriae

✖Blue to green w/ black center

Salmonella typhi Proteus vulgaris Proteus mirabilis

XLD Xylose-lysine deoxycholate

{Selective & Differential agar} Inhibits normal flora coliforms

[Partially E. coli & swarming of

Proteus]

Xylose Na+

Deoxycholate

Phenol red [H2S: ferric ammonium

chitrate; sodium

thiosulfate]

[Lysine AA → Lysine

decarboxylase]

(+)LDC = CADAVERIN

(pH)

✔ Yellow colonies w/ black center (acid)

Citrobacter freundii Proteus vulgaris Proteus mirabilis

✔Yellow colonies w/o black center (acid)

E. coli Klebsiella pneumonia Enterobacter aerogenes Providencia retteri Serratia marcesens

✖Red colonies w/ black center (alk)

Salmonella typhi

✖Red colonies w/o black center (alk)

Shigella dysenteriae

SSA Salmonella-Shigella agar

{Selective & Differential agar} Inhibition of normal flora

coliforms Inhibits Gram (+) & (-)

Lactose Bile salts Brilliant green

Neutral red [H2S: ferric ammonium

chitrate; sodium

thiosulfate]

✔Pink to red colonies (LF)

Normal flora coliforms *Shigella sonnei *Salmonella arizonae (w/ black center)

✖Yellow to colorless w/o black center

Shigella dysenteriae Shigella flexneri Shigella boydii

✖Yellow to colorless w/ black center

Salmonella typhi

TCBS Thiosulfate Citrate Bile salt Sucrose agar

{Selective & Differential agar} Isolate Vibrio sp.

Sucrose

Sodium citrate Sodium

thiosulfate Oxgall

Bromthymol blue

[H2S: ferric ammonium

chitrate; sodium

thiosulfate]

✔Yellow colonies V. cholera V. alginolyticus

✖Blue-green colonies 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

Corynebacterium diptheriae

CYSTEINE TELLURITE AGAR (CTA) >gray to black colonies MODIFIED TYNSDALE AGAR >black colonies with brown halos LOEFFLER SERUM SLANT >enhance METACHROMATIC granules

Francisella tularensis BLOOD CYSTEIN GLUCOSE AGAR

Legionella pneumophilia BUFFERED CHARCOAL YEASE EXTRACT (BYCE) With L-cysteine

Clostridium difficile CYCLOSERINE CEFOXITIN FRUCTOSE AGAR (CCFA)

Prevotella melaninogenica (Brucella) LAKED KANAMYCIN VANCOMYCIN 5% SHEEP BLOOD AGAR (LKV)

5-6 wks: LEPTOSPIRA 3-4 wks: BRUCELLA 2-3 wks: MYCOBACTERIUM

BIPHASIC (2 phased media) >SEPTI-CHECK & OLD CASTAÑEDA *ideal for airborne microbes

BACTERIOLOGY 231

TUBE MEDIA

MEDIA FORMATION CARBOHYDRATE pH INDICATOR H2S INDICATOR RESULT

TSI Triple Sugar

Iron agar Butt Slant

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

Phenol red Ferrous

ammonium sulfate

A/AG E. coli Enterobacter aerogenes Pantoea agglomerans Klebsiella pneumonia Serratia Salmonella arizonae

Decarboxylation (slant) = Alkaline (red)

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

A/AG+ or K/AG+ Citobacter freundii Salmonella arizonae Hafnia alvei Morganella morganii Morganella freundii

A/AG or K/AG Citrobacter koseri Serratia marcescens Hafnia alvei

K/AG+ Proteus vulgaris Proteus mirabilis Salmonella paratyphi Salmonella choleraesuis Edwardsiella tarda

A/A or K/A Yersinia entericolitica Shigella sonnei

K/A Shigella dysenteriae Shigella flexneri Shigella boydii

K/A+ Salmonella typhi

K/K Pseudonmonas aeroginosa Alcaligenes

MEDIA FORMATION AMINO ACID CARBOHYDRATE pH

INDICATOR H2S

INDICATOR RESULT

LIA Lysine Iron

Agar Butt Slant Lysine Glucose

Bromcresol purple

Ferric ammonium

citrate K/K

E. coli Enterobacter aerogenes Klebsiella pneumonia Serratia marcescens Hafnia alvei

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

K/A

Pantoea agglomerans Citobacter freundii Shigella dysenteriae Shigella flexneri Shigella boydii

R/A *deaminase(+)

Proteus Providencia Morganella

K/AG+

Salmonella typhimurium Salmonella arizonae Salmonella typhi Salmonella parayphi Salmonella choleraesuis Edwardsiella tarda

MEDIA PRINCIPLE FORMATION MEDIUM REAGENT POSITIVE NEGATIVE

Indol Production

Test

The organism oxidized tryptophan

by trytophanase ↓

tryptophan ↓

pyruvic acid ↓

amino acid ↓

indol

Butt Tryptone

broth

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.

Red Ring Yellow/Brown

Ring

Methyl Red Test

MIXED ACID

PATHWAY

The organism ferments glucose to

pyruvic acid resulting in mixed acid

products leading to decreased pH (4.5-5.5) of the medium and takes the color

of the indicator

Butt

MRVP (Methyl

Red Voges Proskauer)

medium

Methyl red indicator

Red solution

Yellow solution

Voges Proskauer

Test

BUTYLENE GLYCOL

PATHWAY

The organism ferments glucose

producing acetylmethylcarbinol

(acetoin) and converted to

dimethyl/diacetyl in the presence of

oxygen and KOH.

KOH → reacts w/ GUANIDINE = color

Butt MRVP

VP Reagent A: Alpha napthol in absolute methyl

alcohol

VP Reagent B: 40% KOH with 0.3%

creatine

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 (Semi-

solid)

H2S: ferric ammonium chitrate; sodium thiosulfate

Motile (growth

pattern – away from inoculation

line)

Nonmotile (growth

pattern is confined)

H2S production (Blackening of the butt)

Indol production (red ring)

Brilliant Green Bile

Lactose Broth

Water Analysis Testing >sanitary supply of water

>suitability for general use

Method: Multiple Fermentation Tube Technique

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

COMPLETED TEST: LACTOSE BROTH

Uninocculated

Positive E. coli + Gas Production

+ Turbidity