02 M301 Spec Exam&Cult 2011 - Cal State LA
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Transcript 02 M301 Spec Exam&Cult 2011 - Cal State LA
Specimen Cultivation
How do you grow these bugs?
Clinical Microbiology
Diagnosis
“Gold Standard” – culture isolation and
identification of viable pathogen
Future trend - rapid, non growth
dependent detection of MO
Microbial antigen (specific antibody, i.e.
ELISA)
Microbial nucleic acid - sensitive, need 1 copy,
amplification of DNA, i.e. PCR; RT-PCR (RNA
reverse transcribe to DNA)
Diagnosis: Require Pure
Culture
Identify MO by biochemical methods
Antimicrobial sensitivity testing
In the clinical lab - streak original sample
out on primary isolation media to get
isolated colonies
MO Isolated
Each isolated colony pure culture,
progeny of single cell
Each bacteria forms a characteristic
colony: shape, size, color, texture,
adherence to medium
Colonial characteristics often used as
first step in identification of
bacteria
Bacterial Colonial
Morphology
Culture Media
Artificial media on which bacteria
and fungi grown
Some bacteria never successfully
grown on artificial lab media
Treponema pallidum – grown in testes of
rabbits
Mycobacterium leprae – grown in
armadillo or foot-pad of mice
Obligate Intracellular Parasite
Some bacteria – Rickettsia, Chlamydia
All viruses (non-cellular)
Cultured in living host - whole organism or
tissue cell culture
Identify virus grown in tissue cell
culture by:
Electron microscope - see virus in cell
Cytopathic effects (damage or change in
cell)
Inclusion body (viral products)
Syncytia formation (cell fusion)
Rounding up of cell (death)
Cytopathic Effect: Inclusion
Body (Rabies virus)
Cytopathic Effect: Syncytia
Formation (Herpesvirus)
Cytopathic Effect: Cell
Rounding (Poliovirus)
Diagnosis: Obligate
Intracellular Parasite
Growth time consuming and expensive
Detect MO by immunological methods
Direct assay - antigens of MO, usually in
tissues (use specific antibody as a
reagent)
Indirect assay - host immunological
response (antibody) against MO (use
specific microbial antigen as a regent)
Diagnosis: Antibody Assay
Serology – diagnose infection by assaying
for antibodies in patient’s serum against
causative agent:
Take both acute (patient most ill) and
convalescent (patient recovering) phase
specimen
One looks for a 4-fold rise in antibody titer
between acute and convalescent specimens
ELISA – Enzyme linked immunosorbent
assay:
Use microbial antigen (specificity)
Enzyme detection (sensitivity)
Rapid testing
Diagnosis: Parasites
Not easily grown on artificial media
or in living hosts
Detection based on visual microscopic
identification of:
Parasite (trophozoite, free-living stage)
Ova or cyst stage of parasite
Entamoeba histolytica
(ameba): Trophozoite
Entamoeba coli (ameba):
Cyst
Taenia solium
(tapeworm): Scolex
Taenia solium
(tapeworm): Proglottids
Enterobius vermicularis
(roundworm): Ova
Ascaris lumbricoides
(roundworm): Ova
Culture – When?
Specimen plated immediately
Delay may result in:
Loss of fastidious or anaerobic MOs
Overgrowth by normal flora - change of
total number and relative number of
MOs
Culture Media
Diagnostic labs vary in choice of routine
plating media used for growing different
types of specimens
Take into account what pathogens
anticipated in specimen
Also consider:
Growth requirements
CO2
Temperature requirements (RT, 37ºC)
General Purpose Culture
Plating Media
Supports growth of most common
pathogens, non-selective
Permits isolation and differentiation
of wide variety of bacteria
Differences: colony size, shape,
color, texture, adherence to culture
media
Nutrient Agar Plate
Columbia Blood Agar (CBA)
Plate: Differential
Allows differentiation
based on bacteria
hemolysin that destroy
red blood cells in the
agar
Alpha () hemolysis incomplete hemolysis and
appears as green halo
surrounding the colony
Beta () hemolysis complete hemolysis and
appears as clear area
surrounding the colony
Gamma () hemolysis - no
hemolysis
Chocolate Agar (Choc) Plate
Essentially same as blood agar, except
RBCs lysed
Releases hemin and NAD for fastidious
MOs
Gives medium chocolate brown color
CBA and Choc termed “enriched media”
because of blood nutrients in media
Selective Media
Special nutrients that support growth of
certain pathogens and/or inhibitors that
suppress growth of competing NF
Columbia Blood agar with antibiotics
(Columbia CNA) – select for G(+):
Sheep blood
Antibiotics Colistin & Nalidixic Acid
Why does it inhibit G(–) bacteria?
MacConkey, Salmonella-Shigella, Hektoen
Enteric agar – select for G(-)
Bile salts to inhibit MOs
Why does it inhibit G(+) ?
Selective/Differential Media
Selective media is also usually
differential
Addition of a carbohydrate (CHO) and
a pH indicator differentiate MO that
ferment the CHO and those that do
not
Mannitol Salt Agar (MSA):
Selective - 7.5% NaCl to suppress MOs not
halophilic
Differential - Mannitol (CHO) and pH indicator
phenol red
MSA Plate
MO grows on
media ferments
mannitol, acid is
produced and
lowers pH
At low pH, phenol
red = yellow
MO that
ferments
mannitol turn
media yellow
MacConkey Agar (Mac)
Plate
Selective - Crystal violet and bile
salts inhibit G(+) bacteria, fungi
Differential – Lactose, pH indicator
neutral red (red or pink at acid pH)
Mac plates example of enteric agar
plates which facilitate isolation and
differentiation of enteric pathogens
Mac Agar Plate
MO able to grow on media
and ferment lactose
produce pink colonies
(acid pH) i.e. E. coli
MO that grow and don’t
ferment lactose produce
colorless colonies (neutral
pH) i.e. Salmonella,
Shigella
Reducing Media
Used for cultivating anaerobes
Contains compounds that chemically
combine with dissolved oxygen in media to
deplete O2
Sodium thioglycolate broth:
Thioglycolic acid - reducing agent to create
anaerobic atmosphere deeper in tube
Resazurin - oxygen-reduction indicator; in
presence of O2, resazurin = pink
Growth of MOs in
Thioglycolate Broth
Enrichment Media
To prevent missing bacteria present in small
number
Usually liquid, provides nutrients and
environmental conditions favor growth of one type
MO while unsuitable for others
Enrich stool culture for enteric pathogens found
in low numbers relative to NF:
Gram negative broth - bile & citrate salts inhibit G(+);
mannitol enrich for Salmonella, Shigella)
Tetrathionate broth - bile salts, thiosulfate,
tetrathionate inhibit most G(+) & G(-) rods, except
Salmonella
Selenite broth - selenite inhibits G(-) rods, enterococci;
allows recovery of Salmonella, Shigella
Blood Culture
Collected specimen inoculated into blood
culture media directly at bedside of patient
Two bottles of liquid media inoculated:
Aerobic growth - Tryptic Soy Broth (TSB)
Anaerobic growth – Thioglycolate Broth
Bottles examined for turbidity, 7-14 days
If turbidity develops, some media
removed for Gram staining and subculture
onto solid media
Blood Culture
Blood culture may routinely be
Gram stain and subculture at
specific intervals (24 hrs,48 hrs,
etc.) even in absence of turbidity
Bactec machines automatically
detect growth in blood cultures
by radioactive C14O2 production
Quantitative Culture
Often done on urine specimens
A known volume of specimen plated on agar
medium via calibrated loop and number of
colonies counted
Caution – this represents number of
bacteria present at time of plating
For clean catch urine specimen: >100,000
colonies/ml considered significant and
indicative of disease
For bladder or kidney specimen >10,000
colonies/ml considered significant and
indicative of disease. Why?
Quantitative Urine
Culture Counts
Culture: Unusual MO
Some rarely encountered pathogens
need special media and/or procedure
for isolation
If physician suspects one of these
MO, must notify lab so appropriate
media prepared and proper
precautions taken, if necessary
Brucella
Bordetella
Legionella
Culture Incubation:
Temperature
Inoculated media incubated at 35-370
C, optimum growth temperature for
most human pathogens
Fungi often grown room temperature
Many fungi dimorphic growth:
Yeast at 370 C
Mold at RT
Candida albicans different growth:
Yeast at RT
Mold at 370 C, in the presence of serum (Germ
tube test)
Culture Incubation:
Atmosphere
Most pathogenic bacteria
grow best in 2-10% CO2
Clinical Micro Labs routinely
use 5% CO2 incubators
Some bacteria require 5-10%
CO2 in order to grow or to
grow well (Neisseria,
Streptococcus, Haemophilus)
In the lab, we will grow these
MOs in a “candle jar” to
provide higher CO2 needed
for growth
Anaerobic Culture
Reducing media may be used
Plates may be incubated in special jar or
pouch in oxygen free atmosphere (nitrogen
gas)
In an anaerobic jar, oxygen free
atmosphere generated by chemical
reaction
Anaerobe Jar
Envelopes of sodium bicarbonate and
borohydride placed in jar and water
added
Chemical reaction generates CO2 and
H2
H2 combines with O2 in presence of
catalyst (palladium): 2H2 + O22H2O
Thus O2 removed
Indicator strip – methylene blue:
Colorless in absence of oxygen
Blue in presence of oxygen
Culture Incubation: Time
Most routine cultures: 16-18 hrs
(overnight), before report negative
CSF and blood cultures: one week,
before report negative
Wound cultures: 48 hrs, before
report negative
Fungal cultures: 3-4 weeks, before
report negative
Mycobacterium : 6-10 weeks, before
report negative
Class Assignment
Textbook Reading:
– Chapter 7 Microscopic Examination of
Infected Materials
– Chapter 8 Use of Colonial Morphology for the
Presumptive Identification of Microorganisms
Key Terms
Learning Assessment Questions