Culture of Bacteria

In this article we will discuss about the Culture of Bacteria.

Bacterial Culture:

Bacterial culture provides isolation and identifica­tion of bacterial strains from patients’ specimens, and also for assessing antimicrobial sensitivity. Cultures require a minimum of 12-18 hours of incubation before reports are made.

A. Culture media:

Culture media employed for the isolation of common bacteria are shown in Table 5.2.

1. General purpose media:

Blood agar is the most widely used media, and chocolate agar are used in special cases. Both media support the growth of most organisms. Colonial mor­phology, haemolysis, pigmentation can be determined from the bacterial colonies in blood agar.

2. Special media:

Use of enriched medium (e.g. chocolate agar) facilitates the growth of some organisms (e.g. H. influenzae), selective medium (Thayer-Martin agar) for Neisseria gonorrhoeae, and differential media (e.g. Mac- Conkey’s agar) for culture of gram-negative enteric bacilli.

3. Anaerobic culture media (Robertson’s cooked meat medium) are nutritionally enriched and pre-reduced of molecular oxygen to enhance recovery of anaerobic organisms (Clostridium) from specimens of patients and environment. RCM does not support growth of Non Clostri­dium anaerobes, which requires pre-reduced lacked blood agar supplemented with vit K₁, haemin cystein.

Identification:

The isolated organism is identi­fied by:

(a) Presumptive identification by growth characte­ristics in media, gram stained morphology and motility test, oxidase test and catalase tests.

(b) Results of biochemical tests (carbohydrate utili­sation, indole production, urease production, phenylalanine production, and nitrate redu­ction). Test media are inoculated with bacte­rial culture and incubated for 18-24 hours.

(i) End products of fermentation (acid, gas production) vary with different species of bacteria.

(ii) End products can also be studied by gas- liquid chromatography for identification of non-clostridium anaerobes and myco-bacterium.

(c) Slide agglutination test with high titre sera.

(d) Test for toxin production.

B. Antimicrobial susceptibility testing:

The isola­ted organism is tested for antimicrobial suscepti­bility testing for which the organism must be isolated in pure culture.

Microscopic examination:

A. Unstained preparation:

Unstained preparations (wet mounts) are useful in motility study of bacteria, examinations of deposits of urine, cerebrospinal fluid, faeces and vaginal secre­tions.

B. Stained smears/sections:

Staining is the classic approach for making rapid presumptive diag­nosis:

1. Common stains include Gram and Ziehl-Neelsen stains, and sometimes Giemsa and Methenamine silver-staining.

2. Fluorescent dyes (fluorochromes):

Bacteria stained with fluorescent dye become visible as a bright object against dark background when examined in UV light under a fluore­scent microscope.

(a) Commonly used fluorochromes:

(i) Auramine rhodamine that binds mycolic acid in mycobacterial cell walls.

(ii) Acridine orange, which binds nucleic acid in bacteria and yeast.

(iii) Calcofluor white, which binds cellu­lose fungal cell walls.

(b) Counter-staining:

When examination of stained smear it is necessary.

C. Dark ground illumination (DGI):

DGI, also called dark-field microscopy; uses a dark ground condenser on a microscope that illuminates the object. Bacteria is illuminated over a dark background.

Uses:

This method is extensively used to identify spirochaetes in wet mount, which are too thin to be seen in 1000 x magnification (resolution < 200 nm).

D. Direct immunofluorescence (DIF):

To a tissue or cell suspension, fluorescein-labelled mono­clonal or polyclonal antibodies against specific microbial antigens are added and allowed some­time for reaction to occur to be followed by washing with phosphate buffer. Antigen- antibody complex fluoresces when viewed under fluorescent microscope.

E. Culture, growth character, biochemical tests — are discussed in chapter 6 and 7.

Detection of pathogen-specific molecules:

A. Detection of bacterial antigens is done by:

1. Enzyme-linked immunosorbent assay (ELISA) (Fig. 5.3):

ELISA kits are now commercially available and most tests use antibodies absorbed or cross-linked to a solid phase (e.g. a micro-titre polyvinyl plate, polystyrene tube, a bead or a membrane) that captures antigen in a liquid specimen. The solution to be tested for antigen is placed on the antibody-coated well (or membrane) and incubated.

After the antigen has been bound by the immobilised antibody, an enzyme-linked antibody of the same specificity is added to the mixture and incubated. The antigen, in positive case, is thus sandwiched between the first antibody and the enzyme-linked antibody. Then a specific substrate is added, which produces a visible (coloured) product if the first antibody has successfully captured the antigen.

Uses:

ELISA kits are available for streptococcal antigens, HCG (pregnancy test), toxins (CI. difficile), C. trachomatis and many viruses. These rapid tests require only 10 minutes to 1 hour for completion and easy to perform. Their sensitivity is excellent (greater than 95%).

2. Particle agglutination:

These agglutination tests use antibody-coated latex particles, which are stable for long periods of time. Soluble antigens in the specimen react with antibody-coated particles in the form of cross- linking resulting into visible aggregates.

The Fc regions of IgG antibodies are bound by latex particles or by protein A in killed Staphylococcus aureus, so that the Fab regions remain available for the antigens. These rapid tests are easy to perform and require only 10 minutes to 1 hour for comple­tion. Agglutination assays can be performed with tubes or slides.

Clinical uses:

(i) Latex agglutination test kits are now routinely used for detection of soluble anti­gens in urine, CSF and serum from patients with infections due to Streptococcus pneu­moniae. Haemophilus influenzae, group A, and group B streptococci, Neisseria meningitidis, and Cryptococcus neoformans.

(ii) Slide agglutination tests are also done for rapid identification of organisms grown in culture.

B. Antibody detection in serum:

Qualitative and quantitative Ab titre is detected by whole bacterial agglutination (e.g.. Widal test, Weil-Felix reaction, Brucella agglutination test), Latex agglutination test (e.g.. A.S.O. titre detection), Microscopic Agglutina­tion Test (MAT in leptospirosis), ELISA (e.g. in leptospirosis, Vi-Ag detection), Slide flocculation test (e.g. VDRL test in syphilis), Ag coated R.B.C. agglutination (e.g. T.P.H.A. in syphilis), etc.

C. Molecular detection of microorganisms:

Involve DNA probes and PCR.

1. Nucleic acid probe tests:

Every species of living being has on its chromosome a unique DNA sequence that distinguishes it from every other species. Gene probes are cloned fragments of DNA that recognise complementary sequences of nucleic acid of microorganisms. These are biochemically synthesised or obtained by cloning specific genomic fragments into bacterial vectors (plasmids).

Bacterial restriction endonucleases cleave double- stranded DNA at specific oligonucleotide sequences and produce discrete fragments. These fragments are further separated to single-stranded fragment by electrophoresis.

Tagging probe with marker:

The gene probe thus prepared is tagged or labelled either with radio­isotopes (e.g. ³²P), or an enzyme that gives colour reaction (e.g. alkaline phosphatase) after reacting with substrate.

The single-stranded DNA probe recognises a complementary strand with which it hybridizes under controlled conditions. An instru­ment that measures chemiluminescence emitted by a molecule attached to the probe is used to detect the probe-target hybrid.

Procedure:

In the detection of an unknown micro­organism, its nucleic acid needs to be extracted before they can be hybridized with the probes. The target DNA is readily extracted with the help of NaOH.

There are three formats of nucleic acid hybridi­sation: liquid phase (hybridisation reaction in solu­tion), solid phase (hybridisation in a fixed support such as nitrocellulose or nylon filter, e.g., colony hybridisation), or spot blots, dot blots, or slot blots.

Hybridisation in liquid phase is widely used. When a probe is mixed with test sample, it seeps out and binds to its complementary nucleic acid sequence.

The double-stranded nucleic acid thus formed (capture hybrids) (Fig. 5.1) then gets separated from the rest of the sample. Multiple alkaline-phosphatase conjugates bind to the hybrid amplifying the signal. A chemiluminescent substrate emits light which is measured with a luminometer.

Clinical applications (Table 5.3):

A number of commercially prepared DNA probes for the identi­fication of infectious agents are now available.

(a) DNA probes permit rapid identification of bacteria in patient specimens where reliable culture systems are not practical, such as HPV, hepatitis B virus (HBV), Epstein-Barr virus, N. gonorrhoeae and C. trachomatis.

(b) These are useful culture confirmation probes in problematic cases, such as Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, and Cryptococcus neoformans, Mycobacterium tuberculosis etc.

Other uses of gene cloning:

(a) Production of mammalian proteins, e.g. stomatostatin, human growth hormone, insulin, several interferon’s, and tissue plas­minogen activator.

(b) Preparation of vaccines, e.g. HBV, Rabies, B. pertussis and malaria.

2. Polymerase chain reaction (PCR):

PCR has dramatically changed the technique of detection and characterisation of nucleic acids. The credit for development of this new technology in 1985 goes to its inventor Kary B Mullis (and associates) working at the Cetus Corporation and the Depart­ment of Human Genetics, Emerysville, California. For this discovery, Mullis was awarded the Nobel Prize for Medicine in 1993.

Principle:

PCR is based on the ability of DNA polymerase to synthesise a large amount of specific DNA signature sequence from a single large piece of DNA. Primers required for PCR are usually synthesised using a nucleotide synthesizer.

Technique (Fig. 5.2):

1. Two short DNA primers, which are themselves short complementary oligonucleotides, are selected, which flank the portion of the DNA segment one wishes to identify. A heat-resistant polymerase (taq polymerase, extracted from thermophilic bacterium Thermus aquaticus), and free nucleotides are mixed together. The primers and nucleotides are present in excess.

2. The entire mixture is put into a special heating block (thermo-cycler), which raises and lowers the temperature to permit and regulate the three steps of PCR.

(a) DNA denaturation step:

Two double-stranded DNA is dissociated to a single-stranded DNA at a denaturing temperature between 90°-95°C.

(b) Primer annealing step:

As the mixture cools to approximately -50°-60°C, the primers will anneal wherever they complement the target DNA strands (annealing temperature 50°-70°C). Their presence in large numbers ensures that hybridisation with primers will take precedence over hybridisation with homologous strand.

(c) An extension reaction step:

In this step, temper­ature rises to 72°C (polymerisation temp.), the Taq polymerase adds exogenous nucleotides to the primers, which have hybridized with each original strand.

Elongation — the addition of nucleotides in both the 5′ direction of longer target strand DNA and 3′ direction of short primer— results in the duplication of the target sequence in each chromo­some. Thus, at the end of each cycle, which consists of above referred 3 steps, the PCR products are doubled (i.e. four copies are obtained from the original two).

3. The whole procedure is repeated in a progra­mmable thermal cycler. Generally, 30-50 thermal cycles produce millions of copies of the chosen section of the target organism DNA. PCR is extre­mely powerful and sensitive enough to detect presence of microorganism in a patient specimen.

However, the test should be done carefully, because contamination with microbial DNA from the envir­onment and commensals can lead to false-positive results.

Clinical applications:

PCR techniques are now widely used in the de­tection of infective agents which include:

(a) Viruses: HIV, HCV, CMV, entero-viruses, HBV, HI N1 (swine flu).

(b) Bacteria: Chlamydia trachomatis, Neisseria gono­rrhoeae, Mycobacterium tuberculosis.

RT-PCR:

Reverse transcriptase (RT)-PCR is a variation of the PCR, and it involves the use of reverse transcriptase of retroviruses to convert viral RNA or messenger RNA to DNA before PCR amplification. It has been successfully done with Hantavirus sequences as primers for RT-PCR to detect the agent responsible for an outbreak of haemorrhagic fever in New Mexico. The infectious agent was found to be a Hantavirus.

Detection of proteins:

Some viruses and certain other infectious agents have been identified on the basis of certain characteristic enzymes or specific proteins, e.g. presence of reverse transcriptase enzyme in a specimen (serum, body fluid) or culture indicates the presence of retrovirus.

Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), different strains of virus of bacteria can be distinguished by the pattern of proteins of the concerned species of virus or bacteria.

For example, the electrophoretically separated proteins of HSV will help to distinguish different types and strains of HSV 1 and HSV 2. Specific antibody to the electro­phoretically separated proteins can be used in SDS- PAGE using a Western blot technique.

Serologic testing:

In general, diagnosis of infectious diseases is made on the basis of detection of specific antibody to the infectious agent. The antibody titre reaches detectable level usually after one week of onset of infection and then the titre rises. IgM antibody appears first and IgG antibody appears later.

The following tests are most commonly employed:

1. Agglutination test, e.g. Widal in enteric fever, latex agglutination test in rheumatoid arthritis.

2. Complement fixation test.

3. Flocculation tests, e.g. VDRL and RPR test, routine screening tests for syphilis.

4. ELISA.

5. Indirect immunofluorescence tests, a fluorescein-labelled anti-human immunoglobulin is used to detect specific human antibodies bound to a known antigen, e.g.:

(a) Fluorescent treponemal antibody absorption test (FTA-ABS) in diagnosis of syphilis.

(b) Other commercially available indirect fluore­scent antibody (IFA) test kits include L. pneumophila, Mycoplasma pneumoniae, Borrelia burgdorferi.

6. Immuno-diffusion assays are mainly used for detection of fungal antibodies.