Essential microbiology
BMJ 2004; 328 doi: https://doi.org/10.1136/sbmj.0404142 (Published 01 April 2004) Cite this as: BMJ 2004;328:0404142
Microbiological investigations are crucial to optimally manage infections and infectious diseases. Depending on the results, medical staff can give the appropriate treatment to target the infective organism and minimise the spread of resistant organisms. Staff may also need to initiate public health and infection control measures. Here we provide a brief overview of the techniques available in microbiology laboratories and give examples of their application to different types of specimen.
Specimen collection
You must provide full clinical information on the request form for the laboratory to process the specimen correctly. Microbiologists may process the same specimen type differently in the laboratory depending on the information accompanying the sample. Ideally doctors should not give antibiotics before specimens are taken. However, empirical treatment may be indicated if the patient is seriously ill and waiting to collect specimens would lead to an unacceptable delay. For example, patients with suspected meningitis outside of hospital should be treated with benzylpenicillin.
Take great care when collecting specimens to minimise contamination with environmental organisms. You should put samples in leak proof containers and enclose them in a plastic bag during transport to the laboratory. You should also note any particular infection hazard such as blood borne viruses on the request form, for example HIV, hepatitis B and hepatitis C. If you have any doubt about which investigations are required then discuss the case with the local medical microbiologist.
Principles of investigation
Direct investigations
Direct macroscopic examination of the specimen may be needed. For example, you may see blood in sputum or cerebrospinal fluid may appear cloudy.
Microscopy
Microscopy may involve the use of special stains to identify organisms--for instance, Ziehl-Nielsen stain may be used to identify mycobacteria and Gram staining may be used to classify bacteria according to the composition of their cell walls. Gram positive bacteria include Staphylococcus aureus and Streptococcus pyogenes. Gram negative bacteria include Neisseria meningitidis and Escherichia coli. Other techniques may involve antibodies directed against a pathogen labelled with a fluorescent marker. This technique may be used to identify respiratory syncytial virus.
Culture
Microbiologists may culture organisms in many liquid or solid media depending on the type of specimen and the likely causative organisms. Direct culture of blood confirms the presence of bacteraemia. If an organism is successfully cultured, the type and duration of treatment may be altered after identification of the organism and antibiotic sensitivity testing. Organisms are identified on the basis of morphology, Gram stain, and biochemical tests, such as coagulase and oxidase. Coagulase is an enzyme which converts fibrinogen in plasma to fibrin, thus producing clumping when coagulase positive staphylococci are mixed with plasma. For instance, Staphylococcus aureus has a “bunch of grapes” morphology on Gram stain and is coagulase positive and Escherichia coli is rod shaped and Gram negative.
Most laboratories now use automated systems for culture of organisms. If endocarditis is suspected, you should take three sets of blood cultures to maximise organism yield. In patients with indwelling venous catheters you should take blood for culture both via any lines present and from a peripheral vein.
Serology
Serology is useful in diagnosing viral infections and infections with difficult to culture organisms. Other uses are for screening before vaccination and antenatal screening. Detection of antigens or antibodies in serum may support the presence of infection. Enzyme linked immunosorbent assay (ELISA) is the most commonly used technique, and increasing automation has allowed many samples to be processed rapidly to identify different infectious agents.
The combination of various antibody and antigen assays allows distinction between acute and chronic hepatitis B. For example, if the patient is a carrier there will be persistence of the hepatitis B surface antigen for more than six months and the “e antigen” would be present up to two months after the acute illness, suggesting high infectivity. For many infections detection of IgM is indicative of an acute infection. However, specific IgM assays are not available for all infections and in these cases paired acute and convalescent (when the patient has recovered from the acute phase of illness) sera should be tested; a significant rise in antibody titres is diagnostic.
Molecular
Molecular techniques allow minute quantities of genetic material to be detected by replication or amplification. For instance, HIV viral load can be measured using a polymerase chain reaction to monitor response to HIV antiretroviral therapy. Microbiologists can apply molecular techniques to many types of specimen such as blood, cerebrospinal fluid and sputum. These techniques are of increasing importance in diagnosing meningitis, when prompt antimicrobial treatment has reduced the yield of organisms by conventional culture. Molecular methods are also available for typing or distinguishing between organism subtypes to assess whether transmission of infection has occurred between individuals.
Some infections that may be diagnosed by serology
Syphilis, Lyme disease, toxoplasmosis, Weil's disease, mycoplasma, legionnaires' disease, rickettsia, measles, mumps, rubella, viral hepatitis, HIV, varizella zoster virus, parvovirus B19, and human T cell leukaemia/lymphoma virus
Different specimen types
Sputum
Culturing sputum is useful in the diagnosis of respiratory tract infections. However sputa are often contaminated with upper respiratory tract flora, and results can be difficult to interpret. Samples obtained by bronchoscopic approaches such as bronchoalveolar lavage--in which a known volume of buffered saline is added into the distal airway and then aspirated--are better at predicting the infective organism. However, these techniques are both invasive and expensive and are usually reserved for severe or non-responsive infections.
Other investigations that may be useful include immunofluoresence of sputum for Pneumocystis jiroveci. You may want to try to detect a urinary antigen for Legionella pneumophila or do a high resolution computed tomography scan for Aspergillus lung disease. When you suspect tuberculosis, you should notify the laboratory so that Ziehl-Nielsen staining can be done.
Cerebrospinal fluid
Examination of cerebrospinal fluid is critical for diagnosing meningitis. However, it is vital that investigations do not delay treatment and that doctors give antibiotics and antivirals as soon as possible. When cerebrospinal fluid is taken, cell counts and Gram stains in conjunction with protein and glucose concentrations may give an immediate diagnosis. In addition, you may take throat swabs for bacterial culture, and stool samples for virus isolation, as meningitis may be secondary to infection at these sites. If you strongly suspect infection and results are negative, you can send blood and cerebrospinal fluid for nucleic acid amplification (where genetic material is copied to enable detection). You may also want to send samples for cryptococcal antigen latex agglutination and toxoplasma polymerase chain reaction if the patient is immunocompromised, as this group of patients is more susceptible to such infections.
Urine
To reduce the risk of contamination by perineal organisms, mid-stream urine specimens are needed for microbiological investigation. Microscopy is important for detecting pus cells and bacteria. In asymptomatic, non-catheterised patients, bacteriuria may be defined as more than 105 organisms per millilitre or more than 104 organisms per millilitre in the presence of symptoms, such as increased urinary frequency, dysuria, urgency, and fever. Infection is usually caused by bacteria from the patient's own bowel flora with the most common causative organism being Escherichia coli. Most Gram negative organisms reduce nitrates to nitrites and this forms the basis of a dipstick test that you can perform at the bedside.
Stool
Salmonella sp and Camplyobacter jejuni are the commonest causes of gastroenteritis associated with contaminated food in the United Kingdom. Clostridium difficile toxin may be found in diarrhoea from hospitals' tissue culture techniques. Electron microscopy of the stool sample or an enzyme linked immunosorbent assay (ELISA) can identify rotaviruses and small round structured viruses. Light microscopy is done for ova cysts and parasites--for example, the protozoan Giardia lamblia causing giardiasis and Entamoeba histolytica causing amoebiasis. An accurate travel and food history is important to ensure that specimens are investigated for all likely organisms including those acquired abroad, such as Vibrio cholerae, which causes cholera.
Swabs, pus, and tissue samples
You can swab wounds if you suspect infection. However, taking a sample of pus or liquid is preferable if possible. Wound sepsis is often caused by Staphylococcus aureus and less commonly by Streptococcus pyogenes, the source of infection usually being the patient's own skin or nasal flora. Wound infections after gastrointestinal surgery may be caused by anaerobes and coliforms from the gastrointestinal tract. Infections may be iatrogenic--for example, after inserting intravenous cannulas and central venous lines as well as prosthetic devices. Swabs of the skin may be useful in making a diagnosis of cellulitis and impetigo, since these infections are usually due to Streptococcus pyogenes. A throat swab for group A Streptococcus is usually diagnostic, you would do this in patients presenting with sore throat, dysphagia, and cervical lymphadenopathy. Gram staining of swabs from the genital tract, rectum, and throat may allow a presumptive diagnosis of gonorrhoea to be made; however, confirmation should be got by culture. Different genital infections need swabs from different sites--for example, high vaginal swabs for Candida albicans and bacterial vaginosis, as opposed to cervical and urethral swabs for Chlamydia trachomatis.
Molecular tests available
Blood--HIV, hepatitis C, Epstein-Barr virus, cytomegalovirus, Meningococcus
Sputum--Tuberculosis, pneumocystis, Aspergillus
Cerebrospinal fluid--Meningococcus, herpes simplex virus, enterovirus, Toxoplasma
Genital tract--Chlamydia, Neisseria gonorrhoeae, herpes simplex virus
Other specimens
Doctors may aspirate pleural effusions and send them for microbiological investigation. The exudate may be due to bacterial pneumonia and, less commonly, tuberculosis in the United Kingdom. However, it is important to remember that tuberculosis may be more prevalent in other parts of the world. Ascites is the presence of fluid in the peritoneal cavity, and you should do diagnostic aspiration. Gram stain and culture are helpful if peritonitis is suspected, and cytology with biochemistry is useful to determine if there is peritoneal seeding due to malignancy.
In renal patients with a permanent catheter leading into the peritoneum for continuous ambulatory peritoneal dialysis, peritonitis is a problem, and you should send cloudy fluid from the bags for microscopy and culture.
For suspected septic arthritis, aspiration of the joint space under ultrasound guidance may be diagnostic. Septic arthritis should be suspected if there are signs of inflammation that have appeared acutely or if the patient appears ill. It is most often caused by Gram positive organisms, commonly staphylococci. Septic arthritis is relatively common in children and should be excluded in those presenting with a painful joint.
In addition to fluids, you may send skin scrapings and nail clippings to the laboratory for microscopy and fungal culture if microbiological investigation is helpful in determining a diagnosis.
Microbiological investigations are an essential part of the management of infectious diseases, but to use them effectively it is important to understand a little of the various techniques and procedures available in the microbiology laboratory.
Further reading
Shanson DC. Microbiology in clinical practice 3rd ed. London: Butterworth-Heinemann 1999
Notes
Originally published as: Student BMJ 2004;12:142
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