CLINICAL MICROBIOLOGY

ISOLATION OF MICROORGANISMS FROM SPECIMENS:

FUNGI:

Fungal infections (i.e., mold and yeast infections) often are diagnosed

by direct microscopic (fluorescence) examination of

specimens. For example, the identification of molds often can

be made if a portion of the specimen is mixed with a drop of

10% Calcofluor White stain on a glass slide. Fungal cultures remain

as the standard for the recovery of fungi from patient specimens;

however, the time needed to culture fungi varies anywhere

from a few days to several weeks depending on the

organism. Fungal serology (e.g., complement fixation and immunodiffusion)

is designed to detect serum antibody but is limited

to a few fungi (Blastomyces dermatitidis, Coccidioides immitis,

Histoplasma capsulatum). The cryptococcal latex antigen

test is routinely used for the direct detection of Cryptococcus

neoformans in serum and cerebrospinal fluid. In the clinical laboratory,

nonautomated (conventional kits) and automated methods

for rapid identification (4 to 24 hours) are used to detect

most yeasts. Any biochemical methods used to detect fungi

should always be accompanied by morphological studies examining

for pseudohyphae, yeast cell structure, chlamydospores,

and so on.

 Isolation of Pure Bacterial Cultures from Specimens

Selective Media

A selective medium is prepared by the addition of specific substances to a culture medium that will permit growth of one group of bacteria while inhibiting growth

of some other groups. The following are examples:

Salmonella-Shigella agar (SS) is used to isolate Salmonella and Shigella species. Its bile salt mixture inhibits many groups of coliforms. Both Salmonella and

Shigella species produce colorless colonies because they are unable to ferment lactose. Lactose-fermenting bacteria will produce pink colonies.

Mannitol salt agar (MS) is used for the isolation of staphylococci. The selectivity is obtained by the high (7.5%) salt concentration that inhibits growth of many

groups of bacteria. The mannitol in this medium helps in differentiating the pathogenic from the nonpathogenic staphylococci, as the former ferment mannitol to

form acid while the latter do not.

Bismuth sulfite agar (BS) is used for the isolation of Salmonella typhi, especially from stool and food specimens. S. typhi reduces the sulfite to sulfide, resulting in

black colonies with a metallic sheen.

Differential Media

The incorporation of certain chemicals into a medium may result in diagnostically useful growth or visible change in the medium after incubation. The following are

examples:

Eosin methylene blue agar (EMB) differentiates between lactose fermenters and nonlactose fermenters. EMB contains lactose, salts, and two dyes—eosin and

methylene blue. E. coli, which is a lactose fermenter, will produce a dark colony or one that has a metallic sheen. S. typhi, a nonlactose fermenter, will appear

colorless.

MacConkey agar is used for the selection and recovery of Enterobacteriaceae and related gram-negative rods. The bile salts and crystal violet in this medium inhibit

the growth of gram-positive bacteria and some fastidious gram-negative bacteria. Because lactose is the sole carbohydrate, lactose-fermenting bacteria produce

colonies that are various shades of red, whereas nonlactose fermenters produce colorless colonies.

Hektoen enteric agar is used to increase the yield of Salmonella and Shigella species relative to other microbiota. The high bile salt concentration inhibits the growth

of gram-positive bacteria and retards the growth of many coliform strains.

Enrichment Media

The addition of blood, serum, or extracts to tryptic soy agar or broth will support the growth of many fastidious bacteria. These media are used primarily to isolate

bacteria from cerebrospinal fluid, pleural fluid, sputum, and wound abscesses. The following are examples:

Blood agar (can also be a differential medium): addition of citrated blood to tryptic soy agar makes possible variable hemolysis, which permits differentiation of

some species of bacteria. Three hemolytic patterns can be observed on blood agar;

1. α-hemolysis—greenish to brownish halo around the colony (e.g., Streptococcus gordonii, Streptococcus pneumoniae).

2. β-hemolysis—complete lysis of blood cells resulting in a clearing effect around growth of the colony (e.g., Staphylococcus aureus and Streptococcus

pyogenes).

3. Nonhemolytic—no change in medium (e.g., Staphylococcus epidermidis and Staphylococcus saprophyticus).

Chocolate agar is made from heated blood, which provides necessary growth factors to support bacteria such as Haemophilus influenzae and Neisseria

gonorrhoeae.

Characteristic Media

Characteristic media are used to test bacteria for particular metabolic activities, products, or requirements. The following are examples:

Urea broth is used to detect the enzyme urease. Some enteric bacteria are able to break down urea, using urease, into ammonia and CO2.

Triple sugar iron (TSI) agar contains lactose, sucrose, and glucose plus ferrous ammonium sulfate and sodium thiosulfate. TSI is used for the identification of enteric

organisms based on their ability to attack glucose, lactose, or sucrose and to liberate sulfides from ammonium sulfate or sodium thiosulfate.

Citrate agar contains sodium citrate, which serves as the sole source of carbon, and ammonium phosphate, the sole source of nitrogen. Citrate agar is used to

differentiate enteric bacteria on the basis of citrate utilization.

Lysine iron agar (LIA) is used to differentiate bacteria that can either deaminate or decarboxylate the amino acid lysine. LIA contains lysine, which permits enzyme

detection, and ferric ammonium citrate for the detection of H2S production.

Sulfide, indole, motility (SIM) medium is used for three different tests. One can observe the production of sulfides, formation of indole (a metabolic product from

tryptophan utilization), and motility. This medium is generally used for the differentiation of enteric organisms.

Parasites

Concentrated wet mounts of blood, stool, or urine specimens can

be examined microscopically for the presence of eggs, cysts, larvae,

or vegetative cells of parasites. Blood smears for sporozoan

(malaria) and flagellate (trypanosome) parasites are stained with

Giemsa. Some serological tests also are available.

Bacteria

Isolation and growth of bacteria are required before many diagnostic

tests can be used to confirm the identification of the

pathogen. The presence of bacterial growth usually can be recognized

by the development of colonies on solid media or turbidity

in liquid media. The time for visible growth to occur is an important

variable in the clinical laboratory. For example, most pathogenic

bacteria require only a few hours to produce visible growth,

whereas it may take weeks for colonies of mycobacteria or mycoplasmas

to become evident. The clinical microbiologist as well

as the clinician should be aware of reasonable reporting times for

various cultures.

The initial identity of a bacterial organism may be suggested

by (1) the source of the culture specimen; (2) its microscopic appearance

and Gram stain; (3) its pattern of growth on selective, differential,

enrichment, or characteristic media ; and (4) its hemolytic, metabolic, and fermentative properties on the various media.

After the microscopic and growth characteristics of a pure culture

of bacteria are examined, specific biochemical tests can be performed.

Some of the most common biochemical tests used to identify

bacterial isolates.Classic dichotomous keys are coupled with the biochemical

tests for the identification of bacteria from specimens. Generally,

fewer than 20 tests are required to identify clinical bacterial isolates

to the species level. 

Rickettsias

Although rickettsias, chlamydiae, and mycoplasmas are bacteria,

they differ from other bacterial pathogens in a variety of ways. Therefore

the identification of these three groups is discussed separately.

Rickettsias can be diagnosed by immunoassays or by isolation of the

microorganism. Because isolation is both hazardous to the clinical

microbiologist and expensive, immunological methods are preferred.

Isolation of rickettsias and diagnosis of rickettsial diseases is

generally confined to reference and specialized research laboratories.

Please visit these links: 

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http://www.scienceprofonline.com/microbiology/how-to-isolate-pure-bacterial-cultures-from-clinical-samples.html
http://en.wikipedia.org/wiki/Microbiological_culture

Cited by Kamal Singh Khadka
Assistant Professor in PU, RE-COST, PNC , NA
Pokhara, Nepal