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 + O22H2O
 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