BACTERIOLOGY 231 PLATE MEDIA MEDIA BAP Blood agar CARBOHYDRATE INHIBITOR pH INDICATOR 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Outdated human blood or 5% defribinated sheep blood (preferred) Trypticase Soy Agar or Nutrient agar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 } PINHEAD Staphylococcus PINPOINT - Streptococcus

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

Gamma hemolysis

Nonhemolytic

CAP Chocolate agar

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

{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 (-)

Yellow colonies [pH 6.8]

Coagulase (+) S. aureus Some Micrococcus & Enterococcus

Mannitol

7.5% NaCl

Phenol red

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

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

Beef extract

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) Pink to purple colonies, mucoid

Enterobacter aerogenes (RLF) Motile*

Klebsiella pneumonia (RLF) Nonmotile*

Yellow to colorless (pink) colonies (NLF)

MAC MacConkey agar

{Differential agar} Inhibits Gram Lactose Crystal violet Bile salts Neutral red

Pink to red colonies [pH <6.8]

Yellow to colorless colonies [pH 6.8-8.0]

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

(+) 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] Bromthymol blue [H2S: ferric ammonium chitrate; sodium thiosulfate] Yellow to orange w/ black center Yellow to orange w/o black center

Lactose Sucrose Salicin

Bile salts Citrate

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

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

Phenol red [H2S: ferric ammonium chitrate; sodium thiosulfate] Na+ Deoxycholate [Lysine AA Lysine decarboxylase ] (+)LDC = CADAVERIN (pH)

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

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

SSA SalmonellaShigella agar

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

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

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

Lactose

Bile salts Brilliant green

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

Yellow to colorless w/o black center Yellow to colorless w/ black center Yellow colonies Blue-green colonies

TCBS Thiosulfate Citrate Bile salt Sucrose agar {Selective & Differential agar} Isolate Vibrio sp. Sodium citrate Sodium thiosulfate Oxgall

Sucrose

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

BSA Bismuth sulfate agar

Black colonies with metallic sheen (w/ ring)

Salmonella typhi

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

Bismuth sulfate Brilliant green (Inhibts other enteric)

[H2S: ferric sulfide] soluble

Yellow to orange colonies

S. gallinarum S. choleraesuis S. paratyphi

CYSTEINE TELLURITE AGAR (CTA) >gray to black colonies Corynebacterium diptheriae 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 CASTAEDA *ideal for airborne microbes

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

TUBE MEDIA MEDIA

FORMATION

CARBOHYDRATE

pH INDICATOR

H2S INDICATOR

RESULT A/AG

TSI Triple Sugar Iron agar

Butt Slant

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

Phenol red

Ferrous ammonium sulfate

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

Decarboxylation (slant) = Alkaline (red)

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)

MEDIA LIA Lysine Iron Agar

FORMATION

AMINO ACID

CARBOHYDRATE

pH INDICATOR Bromcresol purple

H2S INDICATOR Ferric ammonium citrate

RESULT 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

Butt Slant

Lysine

Glucose

K/K

K/A

R/A *deaminase(+)

K/AG+

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

MEDIA

Indol Production Test

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

FORMATION

MEDIUM

Butt

Tryptone broth

REAGENT Ehrlichs Add: EtOH and/or XYLENE (xtract indol) then p-dab Kovacs p-aminodimethyl benzaldehyde (p-dab) Spot Indole: rapid detection filter paper:1% pdab in 4 hours.

POSITIVE

NEGATIVE

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 (4.55.5) 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 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. 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.

FORMATION

MEDIUM

INDICATOR

POSITIVE

NEGATIVE

Citrate Utilization Test

Slant

SCA (Simmon Citrate Agar)

Bromthymol blue

Prussian blue color

Green color

Urease Test

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

Brilliant Green Bile Lactose Broth

Positive E. coli + Gas Production + Turbidity