Bacteriology Diagnostic Methods

Abstract

This is summarized in Table 8.1. a) Microscopy— A cell count is performed on sterile fluids and CSF samples using the Neubauer chamber or a similar device. The number of white blood cells (WBC) and red blood cells seen under the microscope are reported as well as the differential WBC count (i.e. the number or percentage of lymphocytes and neutrophils in the sample). A Gram stain is then done and the presence of any organism reported. b) Culture samples are plated onto the appropriate media and streaked out for single colonies as shown. Blood agar is normally used; however, other media are used depending on the site of the specimen, e.g. chocolate agar is used if a fastidious organism is a potential pathogen such as Haemophilus sp.; anaerobic agar for anaerobes; selective agar such as MacConkey can be used on non-sterile specimens to differentiate between the colony types. Plates are incubated for eighteen to forty-eight hours at the correct conditions; most plates being CO2, others at O2 and anaerobically. c) Identification plates are examined for growth. Potentially significant isolates are identified either by MALDI-TOF MS, by API, or other biochemical tests. d) Sensitivities are performed on significant organisms by manual and automated methods. This is summarized in Table 8.2. Selective agar is necessary when isolating pathogens from faeces, although further confirmatory tests are needed. ● Black or colourless colonies on xylose lysine deoxycholate (XLD) or other chromogenic agar plates are tested with oxidase reagent. ● Oxidase negative isolates are identified by MALDI-TOF, API and or biochemically using triple-sugar iron (TSI) tubes. ● Serology is then performed on suspicious isolates and sent to a reference laboratory for confirmation. ● Campylobacter is confirmed by testing grey flat colonies on campylobacter agar with oxidase reagent. Oxidase positive samples are Gram stained and if ‘seagull’-shaped gram-negative bacteria are observed under the microscope, campylobacter is confirmed. The catalase test is a simple biochemical test to differentiate between Staphylococcus species and Streptococcus species, with the use of hydrogen peroxide (H2O2). It tests for the presence of the enzyme catalase which is found in Staphylococcus species.