Enrichment media, either solid or nutrient broth (see Chapter 6 for a broth-screening technique), are generally used for the culture of S. aureus (Health Protection Agency, 2012a). If the specimens have been obtained using charcoal swabs, the swabs are cultured (plated) onto a chromogenic selective MRSA medium and incubated aerobically for 18–48 hours at 37 °C. If nutrient broth has been used, the broth is incubated aerobically for 18–24 hours at 30 °C and then sub-cultured (plated) onto a chromogenic selective medium and incubated for a further 18–48 hours. The culture plates are ‘read’ daily. Individual colonies are 2–3 mm in diameter, circular, with a smooth shiny appearance and a golden-yellow or creamy colour. In order to distinguish MRSA from a sensitive S. aureus, or Staphylococcus epidermidis (coagulase-negative staphylococci), further identification and sensitivity testing need to be undertaken.
Coagulase test
S. aureus has the ability to clot plasma through the production of an extracellular enzyme called coagulase, and the coagulase test (Health Protection Agency, 2010) is the definitive test for dis- tinguishing between S. aureus and other staphylococci. This can be carried out using either a glass slide or a test tube, and involves the use of either human or rabbit plasma. In the slide coagulase test, a drop of distilled water is placed on the slide and the specimen is emulsified to produce a
Box 7.2 Gram-staining technique
The specimen is ‘heat-fixed’ onto a glass slide. Methods of heat fixing may vary but they traditionally involve passing the slide through the flame of a Bunsen burner several times.
The slide is flooded with a blue dye (crystal violet) and left for 30 seconds; iodine is then poured onto the slide to wash away the stain, and then the slide is flooded with fresh iodine and left for 30 seconds.
The iodine is washed off of the slide with either ethanol or acetone until the colour has run out of the slide.
The slide is rinsed with water, and a counter stain is applied by flooding the slide with a counterstain (neutral red, safranin or carbol fuchsin) and leaving it to act for approximately two minutes.
The slide is washed with and then blot dried using blotting paper.
•
Gram‑positive bacteria retain the blue (crystal violet) dye and are stained blue‑black.•
Gram‑negative bacteria are stained red‑pink, as they retain the red safranin dye.Source: Data from HPA, Health Protection Agency, 2011b.
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thick homogeneous suspension. The plasma is added to the slide using an inoculating loop or wire. A positive result sees visible clumping of the cells within 10 minutes. The tube coagulase test gives a positive result in approximately four hours. A test tube containing 1 mL of plasma, diluted according to the manufacturer’s instructions, is prepared, and then a sample from the test strain is added. It is then incubated at 35–37o C and examined hourly for four hours. If a clot forms during that time, the result is positive.
Antibiotic susceptibility
In order to determine the antibiotic susceptibility of organisms and choose the correct therapeutic agents to treat the infection, antibiotic susceptibility and sensitivity testing is required. Isolated colonies are selected from the primary culture plate for testing. The most widely used technique is the disc diffusion method. Using an aseptic technique, an agar plate is inoculated with the test organism and paper discs containing the antibiotic placed on the agar plate using either cooled, flamed forceps or a disc dispenser. The discs are applied no longer than 15 minutes after the agar plate has been inoculated with the specimen, otherwise the organisms may grow, which will affect the zone sizes that form around the discs.
The antibiotic begins to diffuse into the agar immediately, and if it inhibits bacterial growth, a zone of inhibition will become apparent around the antibiotic disc. This means that the organism is sensitive to the antibiotic, and infection can be treated with that antimicrobial agent at the therapeutic dose recommended for treatment of the organism. If it is resistant to the antibiotic, the organism will grow right up to the edge of the disc.
If the strain of S. aureus grown is an MRSA, it will be resistant to the beta-lactam agents (penicillins and cephalosporins). There may be other differences in antibiotic susceptibility which may indicate whether the MRSA is a hospital-acquired or a community-acquired strain.
For example, EMRSA-16, a strain of MRSA common in healthcare settings, is susceptible to ciprofloxacin; community-acquired strains of MRSA are generally ciprofloxacin sensitive. MRSA is discussed in detail in Chapter 20.
Mycobacterium tuberculosis Sputum
The definitive method for detecting whether or not a patient has infectious respiratory tuberculosis (see Chapter 21) is to examine a sputum specimen for the presence of rod-shaped bacilli (Health Protection Agency, 2012b). A mucolytic agent is added to the sputum to break it up, and it is then
‘spun’ or centrifuged to further break down any deposits and leave a ‘pure’ specimen. The bacterial cell walls of Mycobacteria have a high lipid content and generally stain poorly as a result. However, they can be stained through the prolonged application of concentrated dyes, facilitated by heat.
Once stained, the lipids in the cell wall do not dissolve when the stain is washed off with acid–
alcohol, hence the name ‘alcohol–acid fast bacilli’ (AAFB), and they retain the fluorescent stain.
There are two staining methods, auramine–phenol and Ziehl–Nelson (ZN), and the latter is discussed here.
Ziehl–Nelson stain
A thin smear is placed onto a glass slide which is flooded with a strong solution of carbol fuchsin, heated gently until it starts to steam and then left to rest for 3–5 minutes, following which it is
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rinsed thoroughly with water. It is then decolourised for approximately 2–3 minutes with an acid–alcohol solution, which is then rinsed and reapplied for 3–4 minutes until the slide is a faint pink colour. The slide is rinsed thoroughly with water again and counterstained with either methylene blue or malchamite green for 30 seconds, then rinsed with water and allowed to air dry before being examined under the microscope.
If red bacilli are seen against the contrasting background colour, the result is commonly reported as ‘AAFB smear positive’. A diagnosis of ‘open’ respiratory tuberculosis is made if 5000–10 000 acid-fast bacilli are detected in 1 ml of sputum (Pratt et al., 2005; Fitzgerald et al., 2010).
A negative sputum smear does not mean that the patient does not have respiratory tubercu- losis, although it does mean that their infectivity is likely to be low. The sputum specimen will need to be sub-cultured onto special culture media to see if M. tuberculosis can be grown.
The specimen is decontaminated in the first instance to remove any other bacteria or fungi within the sample that might overgrow any Mycobacteria if they are present. It is then used to inoculate the culture media, which are incubated at 35–37 °C for 10–12 weeks and ‘read’ weekly. At the end of the incubation period, the ZN stain is applied to the colonies to detect the presence of AAFBs.
Blood testing
Two blood-based immunological tests are now commercially available in the United Kingdom – QuantiFERON-TB Gold, and TSPOT-TB Assay. These detect tuberculosis antigens known as ‘early secretion antigen target 6’ (ESAT-6) and ‘culture filtrate protein 10’ (CFP-10), interferon gamma produced by T cells in specific response to M. tuberculosis, which are not present in the bacillus Calmette–Guérin (BCG) vaccine and are found in only a few strains of environmental mycobacte- ria. The TSPOT-TB Assay can provide a result within 24 hours.
Clostridium difficile
See Chapter 22 for a more detailed discussion of C. difficile.
Historically, the definitive method and ‘gold standard’ for diagnosing C. difficile infection has been the detection of cytotoxin in tissue culture (National Clostridium difficile Standards Group, 2003). However, revised guidance on the diagnosing and reporting of C. difficile infection was published by the Department of Health on 6 March 2012 (DH, 2012), advising that a two-step approach to testing faecal specimens for C. difficile is adopted. The two tests are GDH (glutamate dehydrogenase) EIA (enzyme immunoassays) or NAAT (Nucleic Acid Amplification Test) or polymer- ase chain reaction (PCR) and a sensitive EIA or cytotoxin assay to detect the presence of toxin gene(s).
GDH is the common antigen seen in all strains of C. difficile. If GDH is detected in a faecal speci- men from a patient with diarrhoea in the absence of toxin, it indicates carrier status. This means that the patient is carrying C. difficile, there is the potential for the patient to develop symptomatic C. difficile infection and therefore there is a risk of cross-infection to other patients. If GDH antigen and toxin are detected and the patient has diarrhoea, then he or she has symptomatic (active) C.
difficile infection. If neither GDH antigen nor toxin is detected, the patient does not have C. difficile, either in a carrier state or as an active infection. See Table 7.1 for a summary of the interpretation of the results arising from GDH antigen and toxin testing.