MICROBIOLOGICAL QUALITY AND SAFETY IN DAIRY INDUSTRY

Module 5. Microbiological quality and safety evaluation of milk and milk products

Lesson 27
ENUMERATION OF STAPHYLOCOCCUS AUREUS

27.1 Introduction

Staphylococcus aureus is highly vulnerable to destruction by heat treatment and nearly all sanitizing agents. Thus, the presence of this bacterium or its enterotoxins in processed foods or on food processing equipment is generally an indication of poor sanitation. S. aureus can cause severe food poisoning. It has been identified as the causative agent in many food poisoning outbreaks and is probably responsible for even more cases in individuals and family groups than the records show. Foods are examined for the presence of S. aureus and/ or its enterotoxins to confirm that S. aureus is the causative agent of food borne illness, to determine whether a food is a potential source of "staph" food poisoning, and to demonstrate post-processing contamination, which is generally due to human contact or contaminated food-contact surfaces. Conclusions regarding the significance of S. aureus in foods should be made with circumspection. The presence of a large number of S. aureus organisms in a food may indicate poor handling or sanitation; however, it is not sufficient evidence to incriminate a food as the cause of food poisoning. The isolated S. aureus must be shown to produce enterotoxins. Conversely, small staphylococcal populations at the time of testing may be remnants of large populations that produced enterotoxins in sufficient quantity to cause food poisoning. Therefore, the analyst should consider all possibilities when analyzing a food for S. aureus. Methods used to detect and enumerate S. aureus depend on the reasons for testing the food and on the past history of the test material. Studies of colonial morphology on Baird-Parker agar, lysostaphin sensitivity, coagulase and thermo-nuclease production, and glucose and mannitol fermentation were conducted on 100 enterotoxaemia and 51 non enterotoxaemia strains of S. aureus. In all cases, the reactions of enterotoxaemia and non enterotoxaemia strains varied by 12% or less. This research indicates that none of these tests can be relied upon to differentiate toxic and nontoxic staphylococci.

27.2 Enumeration Principle of S. Aureus

• This medium contains lithium chloride and tellurite to inhibit the growth of accompanying microbial flora, whereas pyruvate and glycine selectively stimulate the growth of staphylococci
• Staphylococcus colonies show two characteristic features when grown in this opaque medium (opaque, because of its egg-yolk content)

a) characteristic zones and rings are formed as a result of lipolysis and proteolysis,
b) Reduction of tellurite to tellurium produces a black colouration.

• The egg-yolk reaction and tellurite reduction are usually found to occur together with a positive coagulase reaction recommend that egg-yolk should be replaced with blood plasma, if coagulase-positive staphylococci are to be detected directly recommend the addition of sulphamethazine to suppress the growth and swarming of Proteus species.Coagulase in an enzyme capable of coagulating blood plasma.
  

• The coagulase formation by S. aureus and its formation of enteric toxins are very closely related. The Coagulase test is an important indicator for the pathogenicity of Staphylococcus strains. S. aureus forms two kinds of coagulase. Free coagulase is an extracellular enzyme which reacts with a coagulase-reacting factor in plasma (pro-thrombin or its derivatives). Bound coagulase, also known as clumping factor, is localized on the surface of the cell wall and reacts with a- and ß-chains of the plasma fibrinogens to form a coagulate. Because this coagulase test is carried out in a test tube both forms of coagulase are measured.

27.2.1 Typical composition (g/ litre)

Peptone from casein 10.0; meat extract 5.0; yeast extract 1.0; sodium pyruvate 10.0; glycine 12.0; lithium chloride 5.0; agar-agar 15.0. Also to be added: egg-yolk tellurite emulsion 50 ml; if required, added sulphamethazine 0.05 g/ l.

27.2.2 Preparation

Suspend 58 g in 0.95 litre, autoclave (15 min at 121°C). Cool to 45-50°C, mix in 50 ml egg yolk tellurite emulsion and, if required, 50 mg sulphamethazine/ litre. Pour plates. pH: 6.8 ±0.2 at 27°C. The plates are opalescent and yellowish-brown in colour. The ready-to-use culture medium can be stored in the refrigerator (approx. 4°C) for up to one month.

27.2.3 Typical colony characteristics

Typical S. aureus colonies are convex, irregular shape, black, shiny 1-5 mm in diameter with a narrow, white edge surrounded by a clear zone 2-5 mm wide. Opaque rings within the clear zones only appear after 48 hours of incubation (Fig. 27.6). Opaque zone develop around the colonies after 24 hours. S. epidermis growth occurs sometimes: Very small, brown to neither black, non clear zones. Micrococcus with dark brown, dull, clear zones sometimes appear after 48 hours. Bacillus species are white; yeasts have no clear zones.

Fig. 27.1 S. aureus on baird-parkar agar

27.2.4 Enumeration, isolation and identification of staphylococcus aureus

S. aureus is a facultative anaerobic, Gram-positive coccus, which appears as grape-like clusters when viewed through a microscope and has large, round, golden-yellow colonies, often with hemolysis, when grown on blood agar plates. Some strains of S. aureus also produce an enterotoxin that is the causative agent of S. aureus gastroenteritis. Colonies of S. aureus are circular, smooth, convex, moist, 2-3 mm in diameter on uncrowned plates, gray to jet-black, frequently with light-coloured (off-white) margin, surrounded by opaque zone and frequently with an outer clear zone.

27.2.4.1 Media and reagents

Use hydrogen peroxide, Baird-Parker medium & Buffer peptone water for enrichment of sample.

27.2.4.2 Sample preparation

Using aseptic technique, weigh 25 g of sample into sterile blender jar or macerated with sterile mortar pastel. Add 225 ml Butterfield's phosphate-buffered dilution water (1:10 dilution) and mixed well for 2 min. using the 1:10 dilution, make serial dilutions of sample for enumeration of S. aureus.

27.2.4.3 Direct plate counts

For each dilution to be plated, aseptically transfer one ml sample suspension to 3 plates of Baird-Parker agar, distributing one ml of inoculum equitably to 3 plates (e.g., 0.4 ml, 0.3 ml, and 0.3 ml). Spread inoculum over surface of agar plate, using sterile bent glass streaking rod. Retain plates in upright position until inoculum is absorbed by agar (about 10 min on properly dried plates). If inoculum is not readily absorbed, place plates upright in incubator for about one hour. Invert plates and incubate 45-48 hours at 35°C. Select plates containing 20-200 colonies, unless only plates at lower dilutions (>200 colonies) have colonies with typical appearance of S. aureus. Select > 1 colony of each type counted and test for coagulase production. Add number of colonies on triplicate plates represented by colonies giving positive coagulase test.

27.3 Coagulase Test (Clotting of dilute mammalian blood)

27.3.1 Principle

S. aureus is known to produce coagulase, which can clot plasma into gel in tube or agglutinate cocci on slide. This test is useful in differentiating S. aureus from other coagulase-negative staphylococci. Most strains of S. aureus produce two types of coagulase, free coagulase and bound coagulase. While free coagulase is an enzyme that is secreted extracellularly, bound coagulase is a cell wall associated protein. Free coagulase is detected in tube coagulase test and bound coagulase is detected in slide coagulase test. Slide coagulase test may be used to screen isolates of S. aureus and tube coagulase may be used for confirmation. While there are seven antigenic types of free coagulase, only one antigenic type of bound coagulase exists. Free coagulase is heat labile while bound coagulase is heat stable.

27.3.2 Procedure

Dense suspensions of Staphylococci from culture are made on two ends of clean glass slide. One should be labelled as “test” and the other as “control”. The control suspension serves to rule out false positivity due to autoagglutination. The test suspension is treated with a drop of citrated plasma and mixed well. Agglutination or clumping of cocci within 5-10 seconds is taken as positive (Fig. 27.2). Some strains of S. aureus may not produce bound coagulase, and such strains must be identified by tube coagulase test.

Fig. 27.2 Coagulase test

27.4 DNAse Test

27.4.1 Principle

Colonies producing DNase hydrolyse the deoxyribonucleic acid (DNA) content of this medium located in their immediate vicinity. If the medium is then flooded and acidified with 1 N HCl (acid kills the viable cells), the DNA precipitates out (turbidity) and clear zones appear around DNase-positive colonies. Some authors recommend instead flooding the medium with toluidine blue solution or the use of DNase test agars containing toluidine blue or methyl green. These dyes form coloured complexes with DNA. Presumptive pathogenic S. aureus shows colour changes on DNA hydrolysis with pink zone or almost colourless on media containing toluidine blue and methyl green respectively. The pathogenic S. aureus can also be differentiated by exploiting the fact that they metabolize mannitol to form acid, in this case mannitol and a pH indicator (phenol red) must be added to the culture medium.

27.4.2 Procedure

1. Inoculate by streaking a pure culture of the organism to be tested onto the surface of the test agar.
2. Incubate under optimal conditions (in the case of staphylococci, 24 hours at 35°C aerobically).
3. When necessary first check the plates for mannitol fermentation, and then carefully flood the surface of the plates with 1 N hydrochloric acid.

27.4.3 Result and interpretation

Before addition of 1 N HCl into the medium look for yellow surrounded by a yellow zone for by mannitol positive S. aureus. After addition of 1N HCl into the medium HCl appears yellow, surrounded by a yellow zone before addition of 1N HCl, it appears well defined, pink or clearer zones for coagulase positive S. aureus otherwise turbid culture medium