The first essential thing for good microbiological diagnosis is that the specimens reaching the laboratory should be well chosen and of good quality.
A specimen that is poorly taken, badly contaminated or unrepresentative of the infected lesion cannot provide accurate information about the pathogens present no matter how advanced the methods applied to its na ysis. The same may be said of grossly haemolysed, rly preserved or ill-timed serum specimens.
Specimens intended for the detection of bacteria may ‘be divided into those from sites that are normally sterile and those from sites at which there is usually a commensal flora. The former may be sub-divided into those from which material can be taken with little or no contamination and those that may become contaminated during collection. .
Venous or arterial blood, cerebrospinal fluid, pus or tissue samples from “closed” lesions, bone marrows and peritoneal, pleura or other aspirates can be obtained with minimal extraneous contamination.
The lower respiratory and urinary tracts should contain few contaminating organisms, but Sputum and urine collected without special precautions will become contaminated respectively with organisms from the upper respiratory tract or the Urethra. Thus the quality of the sample is of great significance in interpreting the results of urine and Sputum cultures.
Poor quality samples, for example, urine that has taken so long to reach the laboratory, in which multiplication has taken place or “Sputum” that consists mainly of Saliva, should if possible be rejected. Where there is no normal flora to confuse the issue and the specimen has been properly collected and transported, the presence of any organism must be considered of possible significance.
In sites with a normal flora, interpretation may be more difficult. Sometimes, the detection of a_ specific pathogen is all that is necessary, e.g. a group – A Streptococus in a throat swab. More often, interpretation requires exclusion of potential pathogens together with a general consideration of the normal flora.
Finally, there are situations in which it may be necessary to examine the “normal flora” in greater detail. In immunosuppressed patients, the dividing line between commensal and pathogen becomes blurred. The amount of bacteriological support needed by departments specializing in the treatment of such patients is often great, and the methods of specimen – processing and of interpreting the results will be quite different from those in use in the general laboratory.
In this situation, good liason between the microbiologist and clinician is particularly important.
Collections And Transport Of Samples
1. The sample must be of good quality and relevant to the infection:
Many microbiological techniques are relatively insensitive. Successful diagnosis needs the maximum number of pathogens with the minimum number of contaminants.
Where relevant, an aseptic technique of collection should be used. Where pus or serous fluid is available a reasonable volume should be sent rather than a swab. Swabs should be put into transport medium to reduce bacterial death during transit to the laboratory.
Where necessary, invasive or operative methods should be used to obtain samples from deep – seated lesions. Modern imaging techniques may help to localize the lesion and guide sampling.
2. The specimen must be properly labelled and sent in a suitable container:
The label on the specimen and the accompanying request form should provide sufficient information to identify the patient and the nature of the material sent.
The form should also indicate the investigation required, the name of the doctor requesting it and the place to which the report should be sent; it should provide sufficient and concise clinical information and desirable recent or current chemotherapy. Finally, any special risk of infection associated with handling the specimen, e.g. with hepatitis B or human immunodeficiency virus (HIV), should be indicated making use of the symbol for this specified in the regulations of the hospital in question Containers of approved design should be used and should be carefully closed before dispatch to the laboratory.
3. Transport of specimens:
some specimens provide a good medium for the growth of non-fastidious bacteria, notably gram-negative aerobic bacilli. In others, some organisms, notably non-sporing anaerobic bacilli and Neisseria spp., survive poorly.
The rapid transport of specimens to the laboratory is thus a matter of importance. If delay is inevitable, a transport medium or a special container to maintain anaerobiosis may be used; alternatively culture medium can be seeded directly from the patient and then sent to the laboratory. Most general purpose transport media are semisolid and contain a reducing agent and limiting nutrients but they do not provide optimal conditions for the survival of all fastidious pathogens from any type of specimen.
When bacterial overgrowth is the main problem, as with urine, temporary storage at 4°C is an acceptable solution, but low temperatures kill some bacteria, swabs should therefore be kept at room temperature while awaiting transport.
Thus, the methods used to mitigate the effects of delay must be “such as would” to the type of specimen and the organism being sought.
A properly collected specimen is the single most important step in the diagnosis of an infection, because the results of diagnostic tests for infectious diseases depend upon the selection, timing, and method of collection of specimens.
Because isolation of the agent is so important in the formulation of a diagnosis, the specimen must be obtained from the site most likely to yield the agent at that particular stage of illness and must be handled in such a way as to favour the agent’s survival and growth.
For each type of specimen, suggestions for optimal handling are given in the following paragraphs and in section on diagnosis by anatomic site below.
A few general rules apply to all specimens:
1. The quality of material must be adequate
2. The sample should be representative of the infectious process (e.g Sputum, not saliva, pus from the underlying lesion, not from its sinus tract, a swab from the depth of the wound, not from its surface).
3. Contamination of the specimen must be avoided by using only sterile equipment and aseptic precautions.
4. The specimen must be taken to the laboratory and examined promptly. Special transport media may be helpful.
5. Meaningful specimens to diagnose bacterial and fungal infections must be _ secured before antimicrobial drugs are administered.
CLINICAL SPECIMENS
Wounds, Tissues, Bones, Abscesses, Fluids
Specimens from tissue biopsies obtained for diagnostic purposes should be submitted for bacteriologic as well as histologic examination. Such specimens for bacteriologic examination are kept away from fixatives and disinfectants, minced and finely ground, and cultured by a variety of methods.
Bacteriologic examination of pus from closed or deep lesions must include culture by anaerobic methods.
Blood
Blood culture is the single most important procedure to detect systemic infection due to bacteria. Contamination of blood cultures with normal skin flora is most commonly due to errors in blood collection procedure. Therefore, proper technique in performing a blood culture is essential.
The following rules, rigidly applied, yield reliable results.
1. Use only sterile equipment and _ strict aseptic technique
2. Apply a tourniquet and locate a fixed vein by touch.
3. Prepare the skin by applying 2% tincture of iodine in widening circles, beginning with the site of proposed skin puncture. Remove iodine with 70% alcohol. After the skin has been prepared, do not touch it except with sterile gloves.
4. Perform veinpuncture and (for adults) withdraw approximately 20ml of blood.
3. Add the blood to aerobic and anaerobic blood culture bottles.
Take specimens to the laboratory promptly, or place them in an incubator at 379°C
Urine
The following steps are essential in proper urine examination.
a. Proper collection of specimen
Satisfactory specimens from males can usually be obtained by cleansing the meatus with soap and water and collecting midstream. Specimens from females can be obtained after spreading the labia and cleansing the vulva. For most examinations, 0.5ml of ureteral urine or 5ml of voided urine is sufficient. Because many types of microorganisms readily multiply, specimens must be delivered to the laboratory rapidly or refrigerated not longer than overnight.
b. Microscopic examination:
1. Place a drop of fresh uncentrifuged urine on a slide.
2. Cover with a coverglass.
3. Examination with restricted light intensity under the high-dry objective of an ordinary clinical microscope can reveal leukocytes, epithelial cells, and bacteria, if more than 105/™ are present. Finding 10: organisms/mil in a properly collected and examined urine specimen is strong evidence of active urinary tract infection.
c. Culture
Culture of the urine to be meaningful, must be performed quantitatively. Properly collected urine is cultured in measured amounts on solid media, and the colonies that appear after incubation are counted to indicate the number of bacteria per milliliter.
Cerebrospinal Fluid (CSF)
Specimens:
As soon as infection of the central nervous system is suspected, blood samples are taken for culture, and CSF is obtained. To obtain CSF, perform lumbar puncture with strict aseptic technique, taking care noi to risk compression of the medulla by too rapid withdrawal of fluid when the intracranial pressure is markedly elevated. CSF is usually collected in 3 – 4 portions of 2 – 5ml each, in sterile tubes.
This permits the most convenient and reliable performance of tests to determine the several different values needed to plan a course of action.
Microscopic Examination:
Smears are made from fresh uncentrifuged CSF that appears cloudy or from the sediment of centrifuged CSF. Smears are stained with gram’s stain and occasionally with Ziehl Neelsen stain, Cryptococci are best seen in India ink preparations.
Culture:
The culture methods used must favour the growth of microorganisms most commonly encountered in meningitis. Sheep blood and chocolate agar together encourage growth.
Procedure for culture for microscopic examination should be provided for almost all bacteria and fungi that cause meningitis.
Respiratory Secretions
1. Throat: Throat swabs must be taken from tonsillar area before ear swab is taken from the posterior pharyngeal wall.
2. Nasopharynx: Specimens from nasopharynx are studied infrequently because special techniques must be used to obtain them. Whooping cough is diagnosed by culture of B. pertussis from a nasopharyngeal swab spevimen or nasal washings.
3. Lower respiratory tract: Bronchial and pulmonary secretions of exudates are often studied by examining sputum.
Meaningful sputum specimens should be expectorated from the lower respiratory tract and should be grossly distinct from saliva.
Culture: The media used must be suitable for the growth of bacteria.
Gastrointestinal Tract Specimens
Feces and rectal swabs are the most readily available specimens. Bile obtained by duodenal drainage may reveal infection of the biliary tract.
Culture: Specimens are suspended in broth and cultured on ordinary as well as differential media (e.g, MacConkey agar, EMB agar) to permit separation of non-lactose fermenting gram – negative rods from other enteric bacteria.