Most pathogenic bacteria
Pre-Clinical Introduction
and Review
• Neutral pH
• Near body temperature (37°C)
– Most incubators at 35°C
– Lower or higher temps for specific bacteria
Clinical Laboratory Science Program
Michele Jurgensmeier MT(ASCP) SM
Atmospheric requirements
1st step in Specimen Processing
• Obligate aerobes – require oxygen
• Obligate anaerobes – no oxygen
• Facultative anaerobes – growth with or
without oxygen present
• What potential pathogens are you
looking for in this specimen?
• What normal flora can be found in this
specimen?
– Common Facultative anaerobes
• Staphylococcus aureus
• Escherichia coli
• 5 to 10% carbon dioxide
1ST Step in Identification
• Perform Bacterial stains
• Specimen or colony stains
– Gram stain (routine stain)
• Crystal violet, iodine (mordant), acetonealcohol, safranin
– Acridine Orange (sterile specimen, low
numbers of organisms)
– Acid fast stain
– Determines
• Media selection
• Atmospheric selection
• Temperature selection
Bacterial stains
• Help in the preliminary identification of
organism
– Guide to identifications tests
• Guide to media selection
• May not be useful for some body sites
– Stool cultures
• Too much normal flora, gram stain is not useful
1
Media selection
1st in Identification
• Gram stain / Growth on selective media
– Once identify type of organism, gram positive or
gram negative, we can begin identification process
– Generally with gram positive the next step is the
catalase test
• Growth on different media and gram
stain useful in preliminary identification
– Gram negative
• Staphylococcus – Catalase positive
• Streptococcus – Catalase negative
• Growth on MacConkey agar
– Generally with gram negative the next step is the
oxidase test
– Gram positive
• Growth on CNA/PEA agar
• Enterobacteriaceae – Oxidase negative
• Pseudomonas – Oxidase positive
• Neisseria and Moraxella – Oxidase positive
Catalase test
• A colony is placed on a
clean glass slide and a
few drops of 3%
hydrogen peroxide are
placed directly on the
colony. A positive
result shows rapid
production of bubbles
Oxidase test
• Add a few drops of oxidase
reagent to a strip of filter
paper.
• Smear well-isolated
colonies on the reagentsaturated paper with a
platinum loop, or sterile
stick.
• Positive reactions turn
violet to purple immediately
or within 10 seconds at
inoculation site.
Common pathogens
Staphylococcus aureus
• White-gold (cream)
in color
• Beta hemolytic
• Catalase Positive
• Coagulase Positive
• Clinical Significance
– Normal flora of skin and
mucous membranes
– Wounds, bacteremia,
skin infections
Common pathogens
Coagulase negative Staphylococcus
•
•
•
•
•
Gray-white in color
Non-hemolytic, beta-hemolytic
Catalase Positive
Coagulase Negative
Clinical Significance
– Normal flora of the skin and mucous
membranes.
– Associated with infection due to the
widespread use of prosthetic devices,
intravascular catheters, prolonged surgical
procedures, and the presence of
underlying disease and the incidence of
immunocompromised hosts
2
Common pathogens
Staphylococcus saprophyticus
•
•
•
•
Catalase Positive
Coagulase Negative
Novobiocin “R” (<16 mm)
Urinary tract infections in
young females
Common pathogens
Streptococcus pyogenes (Grp. A)
•
•
•
•
Catalase Negative
Bacitracin Sensitive
PYR Positive
Clinical Significance
– Pharyngitis
– Post-streptococcal sequelae
• Acute glomerulonephritis
• Rheumatic fever
PYR
Common pathogens
Streptococcus agalactiae (Grp. B)
• Flat, glossy, grayish-white,
translucent, narrow zone
beta hemolysis
• Catalase Negative
• Bacitracin Resistant
• PYR Negative
• Na hippurate Positive
• CAMP Positive
• Clinical Significance
– Urinary tract infections,
– Neonate pneumonia, septicemia and meningitis
Common pathogens
Streptococcus pneumoniae
• GPC in pairs, lancet-shaped
(can have capsules)
• Small, gray, glistening,
alpha hemolysis
• Optochin / Taxo P sensitive
• Bile solubility positive
• Clinical Significance
– Normal respiratory flora
– Community acquired bacterial pneumonia
– Sinusitis, otitis media, mastoiditis, meningitis,
peritonitis, arthritis, conjunctivitis
Common pathogens
Enterococcus species
• Small, cream / white,
smooth
• PYR positive
• Bile esculin positive
• Growth in 6.5% NaCl
• Clinical significance
PYR
– Normal flora of GI tract
and mucous membranes
– Wound and urinary tract infections
Common pathogens
Enterobacteriaceae species
• Identification - Main Characteristics of
all Enterobacteriaceae
– Gram negative rods
– Glucose = Fermenters
– Oxidase = negative (perform on isolates
from BAP)
– Nitrate to nitrite = positive (reducers) –
rare strains negative
3
Common pathogens
Enterobacteriaceae species
• Most commonly encountered GNR’s in
clinical specimens
• Widely dispersed in nature – soil, water,
plants, intestinal & genitourinary tracts of
humans and animals
• May be considered a pathogen in virtually
any infectious disease & potentially
recovered from any clinical specimen
Common pathogens
Escherichia coli
• # 1 etiologic agent of urinary tract infection
• Meningitis, E. coli 0157:H7 (HUS syndrome),
and gastroenteritis
• KIA = A/A with gas (H2S negative) = lactose
fermenter
• Indole = positive
• Citrate = negative
• Abbreviated Identification
–
–
–
–
– Opportunistic infections
– Overt or primary pathogens (usually
intestinal pathogens)
Common pathogens
Genus Klebsiella
•
•
•
•
•
Lactose fermenters
Nonmotile
Mucoid
Resistant to Ampicillin
Klebsiella pneumoniae
Common pathogens
Genus Shigella
• Non-lactose fermenter
• Nonmotile
• All Shigella isolates must be serotyped to
confirm identification
– Latex agglutination test for “O” somatic antigen
detection
– Disease states
• Opportunistic
• Destructive pneumonia with necrosis and hemorrhage
(sputum red or “currant jelly-like)
• Clinical significance -Overt or primary
pathogen
• Klebsiella oxytoca
– Transmission only human to human
– Agent of bacillary dysentery or
shigellosis
– Opportunistic
Common pathogens
Genus Salmonella
• Non-lactose fermenter
• All Salmonella isolates must be serotyped
to confirm identification
– Latex agglutination test for “O” somatic
antigen detection
• Clinical Significance - Overt or primary
pathogen
– Widely distributed throughout nature –
humans and animals
– Salmonella infections result in varying
degrees of gastroenteritis
Beta-hemolytic
MAC - pink precipitate around individual colonies
EMB - green sheen
Indole positive (spot indole test)
Common pathogens
Campylobacter species
• Curved gram negative rod
• Growth at 42°Celsius
•
•
•
•
Oxidase positive
Na hippurate positive
Nalidixic acid “S”
Cephalothin “R”
• Clinical significance
– Most common cause of bacterial gastroenteritis
worldwide
– Enteritis and diarrheal syndromes – crampy
abdominal pain, bloody diarrhea, chills and fever,
normally self-limiting and resolves in 3-7 days
4
Clinical Microbiology
• Aids in Identification
– Colony Morphology
– Gram stain
– Growth on Selective Media
– Initial tests
Source:
• Mahon, C.R., Lehman, D.C. &
Manuselis, G., Textbook of Diagnostic
Microbiology, 3rd Ed., Saunders
Elsevier, 2007.
• Catalase – gram positives
• Oxidase – gram negatives
5