Diagnostic Microbiology Laboratory

-2015-

Microorganism Identification Process

*Lecture 1*

Introduction to Bacterial Identification Accurate and definitive microorganism identification, including

bacterial identification and pathogen detection, is essential for

correct disease diagnosis, treatment of infection and trace-back of

disease outbreaks associated with microbial infections. Bacterial

identification is used in a wide variety of applications including

microbial forensics, criminal investigations, bioterrorism threats and

environmental studies.

Approach to Laboratory Diagnosis

The laboratory diagnosis of infectious diseases involves two main approaches:

o Bacteriological approach; in which the organism is identified by: microscopic observation, macroscopic identification, biochemical reactions and molecular diagnosis techniques.

o Immunological (serological) approach; in which the organism is identified by detection of antibodies against the organism in the patient’s serum.

Cultural Characteristics

Microorganisms may show distinguishing gross morphologies

when cultured on different media.

This macroscopic appearance of bacteria (characteristic growth

patterns which can be observed with the naked eye) is often

used in their identification.

Cultural Characteristics are observed according to

1. Observe the amount of growtho none = 0o slight = +o moderate = ++ o abundant = +++

2. Coloration- Two types of pigmentation may occur:

a. pigmentation occurring within the organism itself; or

b. water soluble pigment that diffuses into the surrounding medium.

Most organisms will lack chromogenesis (pigment production), exhibiting a white, beige, or gray growth. Pigmentation within the organism may be red, yellow, violet, or other colors.

Soluble pigments may be blue, green, yellow, brown, or other colors. Hold the plate up to the light to examine for diffusible pigments. It may be helpful to compare the color of the agar with an uninoculated media.

3. Opacity- Surface growth can be termed as opaque, as transparent,

or as translucent partial transparency) depending on the degree of

growth.

4. Form- The gross or macroscopic appearance of the growth from the

single streak inoculation is described by:

a. filiform - uniform growth along the line of inoculation.

b. echinulate- margins of growth have a toothed appearance.

c. beaded- separate or semi confluent colonies.

d. effuse- growth is thin, veil-like, unusually spreading.

e. arborescent- branched, tree-like growth.

f. rhizoid- root-like appearance

Morphology and staining

There are many different ways to stain bacteria so that they can be more easily visualized under the microscope. Some stains can also be used to identify and classify bacteria.

o 1. Gram stain

o 2. Acid fast Stain

Other stains used to visualize bacterial structure

o Spore stain

o Flagellar stain

o Capsule stain

Biochemical Characteristics

1. Fermetation & oxidation.

2. IMVIC tests.

3. Biochemical tests.

4. API 20 E.

Other techniques

1 .PCR

2 .Phage typing

3 .Other molecular techniques

Serological characteristics

To Identify several strains of the same type of bacteria we need

to perform serotyping of them such as:

1. Widal test

2. Lancefield grouping

3. Protien A latex

4. others

How to handle microbiological sample Some samples will demonstrate microbial growth and require further

laboratory analysis to identify the contaminants. When growth is detected,

the sample should be taken from the clean section of the laboratory to the

live culture section without undue delay.

Subculturing, staining, microbial identification, or other investigational

operations should be undertaken in the live culture section of the laboratory.

If possible, any sample found to contain growing colonies should not be

opened in the clean zone of the laboratory.

Careful segregation of contaminated samples and materials will reduce false-

positive result..

How to handle microbiological sample Soo it necessary to ensure:

1. Proper collection of the sample

2. Adequate amount

3. Requisitions

4. Tightly capped container

How to handle microbiological sample When needed, a written test request must include the following

information:

1. Hospital No.

2. Full name

3. Gender

4. Date of birth

5. Address

6. Social security no.

7. For female pregnant/ lactating

8. Date of illness

9. Signs/ symptoms

10. Date of onset

11. Recent travel history

12. Immunization

Identification of samples

1. Type of specimen

2. Collection date and time

3. Laboratory number

4. Laboratory findings

5. Test requested

6. Ordering physician

Specimen handling and storage

Samples should be delivered to microbiology central processing

area, within the specified period and then CPA will

1. Check requisition for completeness.

2. Store the sample until they are picked up to microbiology staff.

When STAT requests are received, the CPA staff should inform the

microbiology supervisor.

Specimen rejection criteria

1. The information in the label doesn’t match the information on the

request form.

2. The specimen was transported in improper container or at wrong

temperature.

3. Leaking specimen.

4. The quantity of specimen is insufficient.

Comments

1. Blood received in blood culture is unsuitable for fungal

isolation.

2. Saliva is unacceptable for culture.

3. Multiple samples sent with the same request should be

noticed.

Samples not satisfactory for culturing1. Sample in fixative

2. Dried out swab

3. 24hrs urine or sputum

4. Urine still more than 2hrs at room temperature.

5. Antibiotic medicated patient

6. Anaerobic culture for vaginal, cervical, or urine samples

7. Stool samples from more than 5 days inpatient.

Media Classification

Consistency

Solid

Semisolid

Liquid –Broth-

Nutritional component

Simple

Complex

Synthetic or Chemically Defined Media: Exact chemical composition is known

Functional use

Basic-General- media

Selective

Differential

Enriched

Enrichment

Transport

End of Lecture