enrich sample

cultivate organisms present in low numbers

Supports growth of broad range of organisms

Tryptic soy agar (TSA)

Nutrient Agar

Not selective or differentiating

Contains specific nutrients for the growth of particular species

SBA-TSA with 5% sheep RBC added (used for general isolation of organisms directly from specimens)

Chocolate agar- hemolyzed RBCs and supplements

Chocolate agar supports fastidious organisms

N. gonorrheae and H. influenzae

contains hemolyzed RBCs plus supplements

Contains additives that enhance growth of some organisms while inhibiting others

used for contaminated samples

aids in presumptive ID or grouping of organisms based on the appearance of colonies

beta hemolysis: complete clearing of SBA

Streptolysn O: oxygen labile

Streptolysin S: oxygen stable

alpha hemolysis: incomplete lysing of RBCs leads to

greenish discoloration around colonies

gamma: non-hemolytic

Differential AND Selective 

• Selective: inhibits gram positive organisms
• Differential: lactose plus pH color changing indicator
• Lactase fermentation turns colonies magenta
• non-lactose fermenters stay clear

• Selective-eosin and methylene dyes inhibit Gram + organisms
• Differential: lactose fermenters turn dark due to acid production
• E. coli colonies turn green w/sheen because of high amounts of acid produced
• Lactose non-fermentors are colorless

• Selective only: selects for G+ bacteria
• colistin and nalidixic acid inhibit G - bacteria

• Screens for S. aureus

• Differential: mannitol fermentation produces yellow discoloration (S. aureus)

• Selective: high salt concentration selects for organisms that grow in presence of salt (S. aureus)

Staphylococcus

Streptococcus

Enterococcus

What are the characteristics of Staphylococcus?

Aerobe vs. anaerobe

Gram stain

morphology

catalase test

salt growth?

facultative anaerobe

Gram +

Cocci in irregular clusters

catalase +

grow in high salt media (MSA)

Coagulase test

Coagulase positive=S. aureus group

Coagulase negative=S. epidermidis and S. saprophyticus groups

S. epidermidis is Novobiocin SUSCEPTIBLE

S. saprophyticus is Novobiocin RESISTANT

S. aureus mainly on anterior nares

S. epidermidis mainly colonized skin

mostly hemolytic

mod. large

golden yellow/white

Streptococci are catalase -

Staphylococci are catalase +

Latex beads coated with fibrinogen and Ab to Protein A (component of S. aureus cell wall)

Fibrinogen detects S. aureus coagulase

Ab detect protein A in S. aureus

Streak colony on SBA

place 5 ug novobiocin disc on media

resistance indicated by zone of inhibition less than or equal to 16mm

Enteric organisms

Gram - bacilli

grow well on MAC

Biochemically active

ferments glucose and other sugars (sometime with gas)

catalase +

Oxidase -

reduce nitrate->nitrite

Eschericia (coli

Klebsiella

Proteus (mirabilis)

Enterobacter

Serratia

E. coli 

Salmonella

Shigella

Y. enterocolitica (not covered)

Stx1

Stx2

also known as verocytotoxins

ferment lactose

do NOT ferment sorbitol overnight

SMAC colonies are colorless

Indole + (most E. coli are)

What reaction is responsible for indole test?

How do you know if it is indole +?

metabolic degradation of tryptophan

Tryptophanase breaks down tryptophan->indole

blue green color is +

• Used to differentiate other E. coli from O157:H7
• beta-D-glucuronide enzyme in E. coli degrades MUG and produces fluorescence when incubated overnight

O157:H7 isolates are MUG-

Other E. coli are MUG+

Somatic O antigen (cell wall antigen-outer portion of LPS)

H antigen (flagella)

• Serogrouped based on O antigens
• Serotyped based on flagella H antigens
• reported by genus and serotype to state for tracking outbreaks

Non-lactose fermenter (clear on MAC)

Ferments glucose WITH production of H₂S gas

Agglutination of antisera for Salmonella O groups

TSI: blackening and gas bubble in the deep (H2S  production)

No change in slant color (non-fermenter)

Urease - (no pink color)

What are the key biochemical reactions of Shigella?

Lactose fermentation

other Fermentation

Motility

Gas production

Agglutination

Non-lactose fermenter

ferments GLUCOSE without gas

non-motile

biochemically inactive

H₂S negative

Agglutinates with grouping antisera for Shigella

What does a TSI slant look like when inoculated with Shigella?

Urease slant?

Red in the slant (oxidation of peptones)

Yellow in the deep (production of acid due to glucose fermentation)

Urease -

enrichment broth (maximal recovery)

selective media (MAC, HK)

biochemical screening

serogrouping/(serotyping for Salmonella)

Gram -

lactose negative=clear colonies on MAC (no acid)

Selects for enteric pathogens

contains lactose, sucrose and salicin + pH indicator

Fermenters produce yellow colonies

non-fermenters are green/transparent

Na thiosulfate and ferric ammonium citrate indicate H₂S production through black center of colony

What do Salmonella and Shigella look like on HK agar?

What about E. coli?

Salmonella: clear/green with black center

Shigella: clear green

E. coli (non-O157:H7): yellow

Glucose, lactose, and sucros

phenol red pH indicator

Reaction in the deep: anaerobic fermentation (yellow if acid production due to fermentation)

Reaction on slant: yellow if glucose and lactose/sucrose fermenter, reverts to red if oxidation of peptones causes pH increase

Blackening: H₂S production

Gas production: lifting or cracking of media

Positive:media turns hot pink if urea->ammonia+CO₂ 

Negative: no color change, yellow

Beta: S. pyogenes (GAS) and S. agalactiae (GBS)

Alpha: S. pneumoniae and "viridans" strep

Gamma: S. bovis

What are the key characteristics of Streptococci?

Gram

Colony morphology

Catalase

an/aerobic

Gram positive cocci, spherical/ovoid

usually in pairs or chains

Grey/white translucent colonies

Catalase -

Facultative anaerobes

Lancefield System

Detects cell wall polysaccharides A,B,C,F,G

Cell wall lipteichoic acids (D and Enterococci)

Extract antigens enzymatically by incubating for 10min-1hr

use latex agglutination to identify via homologous antisera

S. pyogenes

pharyngitis, impetigo

cellulitis/necrotizing fasciitis

endocarditis, sepsis

scarlet vever

streptococcal TSS

Sequelae: ARF, glomerulonephritis

Determines the activity of prrolidonyl aminopeptidase enzyme

Presumptive ID of GAS and Enterococci

Blood agar with sulfamethoxazole trimethoprom (STX) to inhibit normal flora

Select colonies with beta hemolysis

Catalase -

PYR +

LOS NICU

Transmission from Mother to child during birth

Sepsis, amnionitis, UTI in pregnant women

Colony morphology on SBA: narrow B hemolysis (some are non-hemolytic)

Lancefield grouping (positive for B antigen)

CAMP +

Streak S. aureus down middle of SBA plate

Streak Streptococcus perpendiculart to it

Look for synergistic hemolysis between GBS and S. aureus

characterized by zone of clearer media around points of intersection 

Streptococcus pneumoniae

"viridans" strep

Optochin sensitive

Bile sensitive
Quellung reaction positive 

What syndromes does S. pneumoniae cause?

**Community acquires pneumonia

Otitis media, sinusitis

meningitis, peritonitis, arthritis

Oropharyngeal carriage common

Large zone of alpha hemolysis

colony encapsulation gives "mucoid" wet appearance (can decrease over time)

Prolonged incubation can cause center of colonies to collapse

Pairs of coccobacilli

non-staining halo indicates capsule

Presumptive ID of S. pneumo

Disk with optochin placed on media plated with colony

Zone of inhibition >14mm = presumptive ID of S. pneumo

Addition of 10% Na desoxycholate (bile salt)

activates lytic enzymes in S. pneumo and causes colonies to disappear

alpha or gamma hemolytic

bile esculin and optochin -

C and G: beta hemolytic, bile esculin and optochin -

D: gamna hemolytic, Bile Esculin + optochin -

What characteristics describe Enterococci?

Gram/morphology

hemolysis

catalase

lancefield group

pyr

bile esculin

salt tolerance

Gram + cocci (single, pair or short chain)

Gamma hemolytic

Catalase -

Lancefield group D (but not GDS)

facultative anaerobe

PYR+

Bile Esculin and Salt tolerant