Specimen and Laboratory Diagnostic Techniques | Microbiology

The following points will highlight the eight methods of collection of specimen and laboratory diagnostic techniques. The methods of collection of specimen are: 1. Respiratory Tract Infection 2. Infection of the Intestine 3. Infection of Urinary Tract 4. Meningeal Infection 5. Infection of Reproductive System 6. Wound Infection 7. Infection of the Conjunctiva and 8. Pyrexia of Unknown or Uncertain Origin.

Collection of Specimen Method # 1. Respiratory Tract Infection:

i. Upper Respiratory Tract Infection:

The upper respiratory tract is frequently the site of gen­eral and localised infection involving the mouth, phar­ynx, nose, nasopharynx, larynx, trachea. The primary infection is often of viral origin and secondary bacterial infection is most often due to the potent pathogens resident in the upper respira­tory tract, e.g., pneumococci, streptococci, Haemophilus influenza and staphylococcus.

The most useful method is to take a swab direct from the surface, if exudate, membrane or pus is present, some of this should be sampled. Films may be prepared from the swabs after culture media have been inoculated, or smears may be prepared directly from the membrane, exudate or surface by means of swabs and stained by suitable stains.

Sore Throat:

This syndrome is mostly due to Strep­tococcus pyogenes and is characterised by acute in­flammation of the tonsillar and faucial areas (acute tonsillitis, acute pharyngitis) with or without exudate, which may be loose or adherent. Acute adenitis, si­nusitis, otitis media, rhinitis, laryngitis, tracheid’s, peri­tonsillar abscess (quinsy) may accompany or follow the acute throat infection.

ii. Lower Respiratory Tract Infection:

In the lower respiratory tract (trachea, bronchi, bronchioles and lung tissue) there is often a primary infection by a virus (e.g., adenovirus, myxovirus, respiratory syncy­tial virus, herpes virus, picornavirus) which is followed by a secondary infection by pathogenic meningo­coccus. From the upper respiratory tract and by spread through the blood and lymph channel or very rarely there is direct extension of infection from liver. In health, the mucous membrane of lower respiratory tract is sterile.

The direct examination of the lower respiratory tract infection is by bronchoscopy or by lung biopsy. This is not always possible in the routine diagnosis of the infection. Sputum from patients with acute bronchitis, bronchiolitis and pneumonia should be examined bacteriologically.

In most cases, the sputum may be the mixtures of exudate and the affected mucous membrane or lung tissue and saliva. In separate puru­lent material containing pathogens from saliva, it is necessary to spread the mixture in a petridish and then pick up directly the purulent material with a loop. Another method is the homogenization of the sputum by adding saponin and incubating for half an hour or by shaking the sputum with glass beads or by digesting with pancreatin.

Direct Film:

Direct film is very useful in the diag­nosis of pneumococcal and staphylococcal infection of the lung after Gram’s stain and also of the pulmo­nary tuberculosis after Ziehl-Neelsen’s method of staining for tubercle bacilli. The direct film may not give any indication about the number of viable or­ganisms; besides, the saliva may contain commensal organisms.

Culture:

Sputum is plated on blood agar for aero­bic and anaerobic incubation and on heated blood’ agar under anaerobic condition with antibiotics disks. For differential diagnosis of pneumococci from Strep­tococcus viridians, an optochin disk is placed on the plate. Pneumococci are sensitive to optochin.

Large number of causative organisms are isolated in spe­cific infections with staphylococci, Haemophilus influenza and pneumococci. In case of suspected tuberculosis, sputum can be homogenized and concentrated before culture on Lowenstein-Jensen medium and before guinea pig Inoculation.

Collection of Specimen Method # 2. Infection of the Intestine:

Acute Intestinal Infection:

Various causative microorganisms (bacterial, virus, protozoa) are respon­sible for acute intestinal infection. The most common pathogens are food poisoning, Salmonella enterotoxic strains of Staphylococcus aureus; less common are Clostridium welchii, Clostridium botulinum. The help of the diagnostic laboratory is essen­tial for the diagnosis of the disease and the speci­mens should be sent for the bacteriological exami­nation.

Specimen Collection:

Faecal sample is much better than the rectal swab; but the rectal swabs may be of necessity to collect sample from the contacts of a patient. It is necessary to ensure that the swabs sample the con­tent of the rectum and that they are not merely placed in the anal orifice.

Faeces should be passed into a clean pot which does not contain any antiseptic and the specimens should be collected free from urine and be sent to the laboratory as soon as possible. If delay is anticipated, the specimen should be trans­ported in glycerol-saline because it prevents the overgrowth of the intestinal commensals on any other enteric pathogens.

In case of cholera, the stool can be collected on a clean paper or leaf, never in a bedpan, because of the bactericidal effect of the antiseptic used in disin­fecting the bedpan; Vibrio choleraecan be transported in alkaline peptone water or alkaline taurocholate tellurite liquid medium or sea water or its equivalent (Venkatraman-Ramakrishnan (VR) fluid) to the bac­teriological laboratory for examination of motility “darting motility”— which is a very important aid for quick diagnosis of cholera. In amebic dysentery, the motile vegetative form of Entamoeba histolytica can be demonstrated within few minutes in the freshly passed faecal sample.

Collection of Specimen Method # 3. Infection of Urinary Tract:

Bacteriological examination of the urine is of great necessity in the diagnosis of the urinary tract infection. Besides, the chemotherapy of the proven infection may be controlled by the in vitro antibiotic sensitivity tests.

Specimen Collection:

Universal containers or disposable plastic pots with tight fitting lids may be used to collect the urine specimens. The wide mouthed containers of 12 oz. capacity can be very useful to collect the mid-stream specimens from females.

A mid-stream specimen of urine (MSU) can also be collected from male patients. Formerly, for bacte­riological examination, a catheter specimen of urine (CSU) was always collected from female patients to avoid contamination of the specimen with organisms from ano-genital region. This method is no longer advocated, since the catheterisation may introduce the infection. The patient should pass the urine with the labia separated and the middle of the stream is collected for the examination.

Once collected, the specimen must be trans­ported to the laboratory immediately (without any delay) because the urine is the best culture medium for the rapid growth of many bacteria. If there may be a delay of 1 -2 hours, the specimen can be stored in the refrigerator to check the multiplication of bacte­ria or it may be transported in a container of low tem­perature. Urine may also be preserved by adding bo­ric acid to a final concentration of 1 -8 per cent.

If the urinary tract is suspected for tuberculosis, the first urine passed in the day is the most suitable specimen (early morning urine).Three complete early morning urine samples should be sent to the labora­tory where the microbiological, cultural and animal tests can be performed on the centrifuged deposit.

Bacterial viable counts can be carried out by:

(i) Pour plate method;

(ii) Inoculating the surface of the medium with known volumes of urine. Colony counts are made after incubation for 24- 48 hours.

There are two semi-quantitative methods of culture of urine:

(a) The first in which a loop of standard diameter is used;

(b) The second — the dip slide — in which a thin layer of agar — either as a coating on a slide or in a metal or plastic spoon — is dipped into a fresh specimen of urine.

In the loop method, the loop is charged with the un-centrifuged urine and plated on solid media. After overnight incubation, the plate culture is in­spected for colonies and the results are evaluated by colony counting.

In the dip slide method, the slide or spoon coated with agar is dipped into a fresh specimen of urine, the excess of urine is drained off; the specimen or spoon can be sent in a suitable container to the labo­ratory. Significant bacteriuria (counts greater than 10^s organisms per ml) may sometimes be found in the absence of symptoms or pyuria, in patients who sub­sequently develop symptoms of urinary infection. There is good evidence of frequent association be­tween a symptomatic bacteriuria and pyelonephritis.

Collection of Specimen Method # 4. Meningeal Infection:

Patients suspected of having meningitis should always have a specimen of cerebrospinal fluid (CSF) examined in the laboratory as soon as possible. The causal organism should be identified promptly. It is very important because the proper antimicrobial therapy cannot be prescribed until the exact bacte­riological diagnosis is made.

Specimen Collection:

Cerebrospinal fluid should be collected very carefully by lumbar puncture. It is safe to remove 5-10 ml of CSF as long as intracranial pressure is not increased. One or two sterile screw capped containers can be used to collect the CSF. Test tubes with cotton plugs should not be used because, if they are shaken or fall over, the CSF may be absorbed by the plug.

The specimen should be sent immediately to the diag­nostic laboratory, because meningococci — being delicate — may die, leucocytes may disintegrate and the concentration of sugar in the CSF may be reduced. Specimen should not be kept in the refrigerator which kills H. influenzae .

Examination for Cells:

There is a great increase in the number of leucocytes in the CSF which becomes turbid in acute bacterial meningitis. One mm³ of CSF may contain up to several thousand cells and in early stages of the disease, almost all of them are polymorphs. But there are fewer cells in CSF (200-500 per mm³) with pre­dominant lymphocytes in tuberculosis meningitis. In case of virus infections of the meninges, there are only 50-100 lymphocytes per mm³ of CSF resulting in “aseptic” type of meningitis.

Biochemical Examination:

Quantitative biochemical estimation of protein, sugar (glucose) and chloride content of the CSF should be conducted.

Examination for Microorganisms:

(a) Acute Purulent Meningitis:

In this condi­tion, the CSF is turbid. Neisseria meningitidis, pneumococci, H. influenzae are responsible for this type of meningitis as they may reach meninges via the blood stream, because of their simultaneous iso­lation by blood culture. The infections of middle ear, para-nasal or frontal sinuses may spread directly to the meninges.

Variety of organisms (Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas phyocyanea, Escherichia coli, Bacteroides, anaerobic streptococci) may gain access to the meninges after careless lumbar puncture.

In the laboratory, films made from CSF centrifuged deposit are stained by Gram’s stain and examined. The remaining deposits are cultured on blood and heated blood agar (chocolate agar) medium and incubated anaerobically, in 5-10 per cent CO₂ at 37°C. A primary antibiotic disk sensitivity test is done if large number of organisms is seen in the stained film.

(b) Acute Non-Purulent Bacterial Meningitis:

Mycobacterium tuberculosis or Leptospira icterohaemorrhagiae may cause acute non-purulent bacterial meningitis and the CSF is not usually turbid. A veil clot appears in the CSF on standing, in the case of tuberculosis meningitis and on microscopy by Ziehl-Neelsen’s stain, acid fast bacilli can be demonstrated. CSF centrifuged deposit may be cultured on Lowenstein-Jensen medium and by guinea pig inoculation. Serological tests are useful to diagnose leptospiral menin­gitis.

(c) Viral (Aseptic) Meningitis:

In this condi­tion, the CSF is not turbid. Picorna virus, coxsackie virus, poliovirus (least) are re­sponsible for this infection. Echovirus and coxsackie virus, though they are present in faeces, can be isolated directly from CSF. Hence, CSF and faeces should be sent to the laboratory. These materials can be stored up to 24 hours at 0° – 4°C or frozen at – 30°C or in 50 per cent glycerol saline.

Collection of Specimen Method # 5. Infection of Reproductive System:

Coliform bacilli, streptococci, Mycobacterium tuberculosis, Neisseria gonorrhoeae are pathogens responsible for the infection of female genital tract.

Pueperal sepsis or septic abortions which are acute infections in the female after delivery are caused by Streptococcus pyogenes, Clostridium weichii, Bacteroides, Coliform bacilli.

Cervical swabs should be taken with the help of a speculum under direct vision, put in to Stuart’s trans­port medium and sent to the diagnostic laboratory.

In acute vaginitis due to Trichomonas vaginitis and vaginal thrush due to Candida albicans, exudates can be readily obtained by swabbing and direct smears can be made on slides for microscopic ex­amination or exudates may be collected by pipette or spoon for wet films. Acute urethritis and prostatitis in male are due to N.gonorrhoeae, chlamydia or Mycoplasma hominis.

Collection of Specimen Method # 6. Wound Infection:

Wound infection may be:

(a) Endogenous, or

(b) Exogenous.

Endogenous infections (auto-infection) are caused by organisms that were commensals else­where in the host body, e.g., organisms from large intestine may infect the abdominal surgical wounds after an operation that has involved the incision of the skin. Exogenous infections are due to organisms from outside the host who has become infected.

Cross-infection is an example of exogenous in­fection where the causative organism is spread from person to person. Infection may occur after acciden­tal or intentional trauma of the skin or other tissues: the latter type is known as “post-operative sepsis.”

Specimen Collection:

Swabs should be well soaked in pus or exudate from infected wounds. Syringe can be used to collect the pus or exudate specimens which can be trans­ferred into a sterile test tube. Pieces of tissues re­moved at operation or curetting’s or other tissues can be sent immediately to the bacteriological labora­tory and they should be homogenized in a tissue grinder before being used for bacteriological exami­nation. Delay in the transit of specimens to the labo­ratory must be avoided, particularly in cases of swabs where the exudate may dry in the cotton-wool.

Collection of Specimen Method # 7. Infection of the Conjunctiva:

Pseudomonas pyocyanea causes severe con­junctivitis due to the use of contaminated eye drops. Gonococcus (ophthalmia neonatorum), Staphyloco­ccus aureus (sticky eye in new born babies), pneumococci, Moraxella lacunata and H.influenzae, certain viruses (herpes virus, adenovirus), TRIC agent (Trachoma, inclusion blenorrhea) produce conjuncti­vitis.

Swabs coated with exudates can be sent to the laboratory in Stuart’s transport medium. Purulent exu­date from inflamed conjunctiva can be considered as pus. Gonococcus can be shown by direct microscopy and can be grown on heated blood agar medium.

TRIC agents can be demonstrated in the scrapings taken from the conjunctiva, spread on slides, stained with Giemsa stain and examined for basophilic inclusion bodies or stained by a fluorescent conju­gate antiserum and examined by fluorescent microscopy.

Collection of Specimen Method # 8. Pyrexia of Unknown or Uncertain Origin:

Patients with a significant and persistent fever (greater than 100°F = 38°C) are classified as “Pyrexia of Unknown Origin” (PUO) since the cause cannot be readily diagnosed on clinical examination. PUO may be due to the infection which can be diagnosed by the presence of a specific pathogen in the blood or tissues. In addition to isolation (direct method) of causative agent, the serological tests (indirect meth­ods) can also be performed to demonstrate the spe­cific antibody. The possibility of an exotic infection may be included if the patient was abroad recently.

(i) Examination of Blood:

(a) Blood Film:

The examination of blood films may rule out the presence of malarial parasites (Plasmodia) and reveal the white blood cell picture of the peripheral blood.

(b) Blood culture can be done on suc­cessive days or on several occasions each day or over a period of several hours as in the case of sub-acute bac­terial endocarditis, because of the number of causative organisms which may be very small and of the inhibitory effects of antibiotics on the circulating pathogens and special media (Brucella or Albimi agar) are required for the isolation of Brucella and an atmosphere of 10 per cent carbon dioxide for isolation of Bru­cella abortus. Salmonella typhi can be isolated in a blood culture bottle containing the bile salt broth.

(c) Animal Inoculation:

Leptospira, Bru­cella, Rickettsiae can be isolated from the fresh blood on animal inocula­tion which is more successful than on artificial media.

(d) Examination of Serum:

Part of the blood used for culture may be al­lowed to clot and the serum is used for serology. Serological studies should be done at an interval of 5 to 10 days during the acute stage of the disease and later during the conva­lescent stages to demonstrate rise or fall in the amount of antibody. Examination of paired specimens (5 to 10 ml each) should always be made for antibodies to the enteric group of Salmonella (Widal test) and Bru­cella species.

(ii) Examination of Urine:

Routine examina­tion should be carried out to exclude the infection of urinary tract. In chronic pyelonephritis, Escherichia coli may be present only on intermittent occasions. Salmonella sp. can be isolated occasion­ally from the urine. Repeated examina­tions may be necessary.

In genitourinary tuberculosis, three entire morn­ing specimens or a 24 hour specimen of urine should be collected; pyuria is usually present and Myco. tu­berculosis can be seen after centrifugation and stain­ing of the urine deposit; in leptospirosis, leptospirosis can be demonstrated by dark field examination of the fresh alkalized urine.

(iii) Examination of Faeces:

In intestinal infec­tion, Shigella, Salmonella, intestinal proto­zoa and the ova of helminths can be dem­onstrated in the faeces. In suspected virus infection it is advisable to watch the virus infection with the rise of the homologous antibody titre of the serum.

(iv) Examination of Tissue:

Tissue, such as lymph node, may be cultured for Myco. tuberculosis after grinding it in a homogenizer or tissue blender. Tissue can be collected by biopsy.

(v) Examination of other body fluids can be done by microscopy or by culture. Aspira­tions of secretion of bile in suspected infections of the gall bladder, liver or bil­iary passage, CSF in meningitis and bone marrow in enteric fever are used to con­firm the suspected infection.

(vi) Skin tests may be useful in the diagnosis of sub-acute or chronic infections. The in­oculation of a small quantity of bacterial products, e.g., tuberculin, brucellin, the Frei antigen results in a localised delayed type hypersensitivity reaction.

Body fluids and discharges with infectious or­ganisms and fomites likely to be contaminated: