Specimen CultivationSpecimen CultivationSpecimen CultivationSpecimen CultivationHow do you grow these bugs?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