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 INFECTIOUS DISEASE

BACTERIOLOGY IMMUNOLOGY MYCOLOGY PARASITOLOGY VIROLOGY

VIDEO LECTURE

BACTERIOLOGY - CHAPTER TWO  

CULTURE AND IDENTIFICATION OF INFECTIOUS AGENTS  

Dr Alvin Fox

    
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Logo image Jeffrey Nelson, Rush University, Chicago, Illinois  and The MicrobeLibrary

Reading: Murray, 6thedition, Chapter 3

KEY WORDS

Isolation (culture)
      Agar plate/colonies
      Liquid media - test tube - bulk

Identification & taxonomy
     
Family 
      Genus
      Species 
      Type

Biochemical (physiological) tests

Molecular tests
      DNA-DNA homology
      16S rRNA sequencing

Chemical profiling

Non culture based detection
      Polymerase chain reaction (PCR)
      Agglutination (antigen detection)
      Stain
      Serology (antibody detection

Bacterial identification in the diagnostic laboratory versus taxonomy

Isolation and identification of bacteria from patients aids treatment since infectious diseases caused by different bacteria have a variety of clinical courses and consequences. Susceptibility testing of isolates (i.e. establishing the minimal inhibitory concentration or MIC) can help in selection of antibiotics for therapy. Recognizing that certain species (or strains) are being isolated atypically may suggest that a disease outbreak has occurred e.g. from contaminated hospital supplies or poor aseptic technique on the part of hospital personnel.

When patients are suspected of having a bacterial infection, it is usual to isolate visible colonies of the organism in pure culture (on agar plates), and then speciate the organism. The identification is based on taxonomic principles applied to the clinical microbiological situation. In the diagnostic laboratory, many samples must be characterized each day and results obtained as quickly as possible. Tests must be easily learned, low in cost and rapidly performed. These classical methods for speciation of bacteria are based on morphological and metabolic characteristics. The diagnostic tests have been selected on the basis that empirically they provide discriminating information. There are numerous different tests for each of the many target pathogens. Additionally, molecular biology techniques (for characterization of specific genes or gene segments) are now commonplace in the clinical laboratory.

Modern taxonomic approaches often employ technically more complex methodology and are concerned with profiling the structural composition of bacteria. This often involves "molecular biology" or "analytical chemistry" -based approaches. It is now recognized that many of the classical schemes for differentiation of bacteria provide little insight into their genetic relationships and in some instances are scientifically incorrect. New information has resulted in renaming of certain bacterial species and in some instances has required totally reorganizing relationships within and between many bacterial families. 

blood agar2.jpg (68254 bytes) Figure 1 Bicarbonate and blood agar plate cultures of Bacillus anthracis. Smooth colonies on bicarbonate (left)  and rough colonies on blood agar (right). CDC/Dr. James Feeley 

gram-st.jpg (147324 bytes) Figure 2. The Gram stain procedure

Taxonomic terms (classification)

Family: a group of related genera.

Genus: a group of related species.

Species: a group of related strains.

Type: sets of strain within a species (e.g. biotypes, serotypes).

Strain: one line or a single isolate of a particular species.

The most commonly used term is the species name (e.g. Streptococcus pyogenes  - abbreviation S.pyogenes). There are always two parts to the species name, one defining the genus in this case "Streptococcus" and the other the species (in this case "pyogenes"). The genus name is always capitalized but the species name is not. Both species and genus are underlined or in italics.

 

Steps in diagnostic isolation and identification of bacteria

Step 1. Samples of body fluids (e.g. blood, urine, cerebrospinal fluid) are streaked on culture plates and isolated colonies of bacteria (which are visible to the naked eye) appear after incubation for one to several days (Figure 1). Each colony consists of millions of bacterial cells. Observation of these colonies for size, texture, color, and (if grown on blood agar) hemolysis reactions, is highly important as a first step in bacterial identification. Whether the organism requires oxygen for growth is another important differentiating characteristic.

Step 2. Colonies are Gram stained and individual bacterial cells observed under the microscope.

Step 3. The bacteria are speciated using these isolated colonies. This often requires an additional 24 hours of growth.

 

FIGURE  3. EXAMPLES OF GRAM STAINS

newgram.jpg (25333 bytes)  Bacillus brevis. Gram stain. CDC/Dr. William A. Clark 

newgram2.jpg (157753 bytes)  Streptococcus mutans. Gram stain. Blood agar plate culture yields coccal-like morphology without chains. Organism can cause subacute bacterial endocarditis and dental caries. CDC/Dr. R Facklam 

newgram3.jpg (46817 bytes)  Bacillus anthracis. Gram stain. CDC/Dr. James Feeley 

newgram4.jpg (89145 bytes)  Streptococcus mutans. Gram stain.  Thioglycollate broth culture. Morphology is rod-like with chains when cultured on broth. Can cause subacute bacterial endocarditis and dental caries. CDC/Dr. Richard Facklam 

The Gram Stain

A colony is dried on a slide and treated as follows (Figure 2 and 3):

Step 1. Staining with crystal violet.

Step 2. Fixation with iodine stabilizes crystal violet staining. All bacteria remain purple or blue.

Step 3. Extraction with alcohol or other solvent. Decolorizes some bacteria (Gram negative) and not others (Gram positive).

Step 4. Counterstaining with safranin. Gram positive bacteria are already stained with crystal violet and remain purple. Gram negative bacteria are stained pink.

Under the microscope, the appearance of bacteria is observed. Questions to be asked include: 

  • Are they Gram positive or negative? 
  • What is the morphology (rod, coccus, spiral, pleomorphic [variable form] etc)? 
  • Do cells occur singly or in chains, pairs etc? 
  • How large are the cells? 

Besides the Gram stain, there are other less commonly employed stains available (e.g. for spores and capsules).

Another similar colony from the primary isolation plate is then examined for biochemical properties; for example, will the bacteria ferment a sugar such as lactose? In some instances, the bacteria are identified (e.g. by aggregation) with commercially available antibodies recognizing defined surface antigens. Other commercial molecular tests are now widely used.


Taxonomic characterization of bacteria

There is considerable diversity even within a species. Thus comparisons of species involve comparisons of multiple strains for each species. Comparisons are primarily based on chemical or molecular analysis.
 

Chemical analysis
Sophisticated tools are available for studying the structural composition of bacteria (most commonly fatty acid, carbohydrate or ubiquinone profiling). Characterization of secreted metabolic products (e.g. volatile alcohols and short chain fatty acids) is also helpful.
 

Molecular analysis 
It would be ideal to compare sequences of entire bacterial chromosomal DNA, but this is currently not feasible. Millions of nucleotides have to be sequenced for each strain. In the past several years, sequencing of the entire genomes of one representative (i.e. a strain) of a few bacterial species has been achieved. In each case, this has involved massive amounts of work by large research groups dedicated to the task of sequencing. Alternatively, genomic similarity has been historically assessed by the content of guanine (G) plus cytosine (C), usually expressed as a percentage (% GC). This has been replaced by two alternatives - hybridization and sequencing (most commonly of the gene coding for 16S rRNA).

DNA-DNA homology (or how well two strands of DNA from different bacteria bind [hybridize] together) is employed to compare the genetic relatedness of bacterial strains/species. If the DNA from two bacterial strains display a high degree of homology (i.e. they bind well), the strains are considered to be members of the same species. DNA from different bacterial species (unless closely related) display no homology.

In the last few years, sequencing of 16S ribosomal RNA molecules (16S rRNA) has become the "gold standard" in bacterial taxonomy. The molecule is approximately sixteen hundred nucleotides in length. The sequence of 16S rRNA provides a measure of genomic similarity above the level of the species allowing comparisons of relatedness across the entire bacterial kingdom. Closely related bacterial species often have identical rRNA sequences. The technique thus provides complementary information to DNA-DNA hybridization.  Determinations of the sequence of 16S rRNA genes and other genetic regions are used in identification in the clinical microbiology laboratory.
 

Approaches to rapid diagnosis without prior culture

Certain human pathogens (including the causative agents of tuberculosis, Lyme disease and syphilis) either cannot be isolated in the laboratory or grow extremely poorly. Successful isolation can be slow and in some instances impossible. Direct detection of bacteria without culture is possible in some cases.

A simple approach to rapid diagnosis (as an example of antigen detection) is used in many doctor's offices for the group A streptococcus. The patient's throat is swabbed and streptococcal antigen extracted directly from the swab (without prior bacteriological culture). The bacterial antigen is detected by aggregation (agglutination) of antibody coated latex beads.

Bacterial DNA sequences can be amplified directly from human body fluids (the polymerase chain reaction, PCR). In this fashion large amounts of specific genes or portions of genes can be generated and readily detected. For example, great success has been achieved in rapid diagnosis of tuberculosis.

Finally, direct microscopic observation of certain clinical samples for the presence of bacteria can be helpful (e.g. detection of M. tuberculosis in sputum).

Serologic identification of an antibody response (in patient's serum) to the infecting agent can only be successful several weeks after an infection has occurred.
 

 

 

 

 

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