2 - NUTRIENT BROTH CULTURE This is a simple test to determine whether an organism will grow in nutrient broth.

The medium is inoculated with a speck of culture using a sterile loop. If the culture has not grown, the broth should be crystal-clear [1]. Growth is indicated by turbidity after incubation [2], but make sure the bottle is agitated before you examine the broth for turbidity to resuspend any cells that have fallen to the bottom. Some microbes may produce a pigment [3], while others (such as Bacillus species) may produce a skin (pellicle) on the surface of the liquid [4]. Look for these possibilities.

3 - NUTRIENT AGAR STREAK PLATE The streak plate technique is a means of achieving cell separation when inoculating the surface of an agar plate, so that, after incubation, the resulting colonies grow separately from each other. This enables colony features to be described on the basis of shape, size, colour and finish (mat, lustre, glossy) - all of which are used when identifying an unknown culture. A streak plate is also useful for purifying a mixed culture since the separate colonies can be sub-cultured onto new media and grown as pure isolates. There are various ways of making a streak plate but the one described here is as good as any. The agar surface must be dry so the plate is first over-dried and then a speck of culture is streaked with a loop, using only light pressure, onto the agar surface along one side of the plate [from direction 0 to 1 in the photo below]. The loop is then flame-sterilised, allowed to cool and used to streak material from from the end of the first set of streaks in a new direction [1 to 2 in the photo]. The loop is again flamed and cooled and a third set of streaks made [from 2 to 3 in the photo] using material from the end of 2nd set of streaks. The loop is again flamed and cooled and a final set of streaks made into the centre of the plate [from 3 to 4 in the photo] using material from along the length of the 3rd set of streaks. In this way, the culture has been 'thinned out' so that isolated colonies grow after incubation. You can see how all this is achieved by watching the video below.

Making a streak plate (video duration 2' 42")

4 - NUTRIENT AGAR STAB

The agar stab technique involves using a straight wire (rather than a loop) to inoculate a well-filled bottle of agar medium which has been allowed to set upright (rather than in the sloped position). Such a medium is sometimes referred to as an agar deep. The stab technique allows aerobes to grow at the top of the stab line, while organisms with anaerobic tendencies will grow along the stab line, without profuse growth at the agar surface. To make a nutrient agar stab culture, a straight wire is first flame-sterilised and allowed to cool. It is then rolled onto a slope culture or immersed in a broth culture to pick up some cells. The wire is then stabbed down the centre of the nutrient agar until the wire hits the bottom of the bottle. The position of the growth along the stab line after incubation will give an indication of the oxygen requirements of the organism. Microbes are classified as aerobic if they require oxygen for growth, anaerobic if they cannot grow in the presence of oxygen facultative if they can grow in either situation.

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5 - GELATIN STAB Some microbes secrete gelatinase which hydrolyses the protein gelatin, causing it to liquefy. Other microbes do not possess this extracellular enzyme and are unable to liquefy gelatin so this test is useful in identification. The bottles of nutrient gelatin are inoculated by the stab technique (see NA stab) and incubated at 37 C. At this temperature gelatin is liquid (its setting point is about 25 C) so, after incubation, the cultures have to be cooled before they can be examined for liquefaction. In a positive result, the gelatin will be liquid after cooling (right hand bottle in photo) in a negative result, the gelatin will have set (left hand bottle in photo).

6 - THIOGLYCOLLATE STAB Sodium thioglycollate is a reducing agent and its incorporation into a medium makes it suitable for growing aerobic bacteria at the top, and anaerobic bacteria at the bottom, of a well-filled bottle (i.e. it is a dual-purpose medium). The medium is inoculated by the stab technique (see NA stab) and the position of the growth observed after incubation to determine the oxygen requirements of the organism. Because the medium is fluid, it is important not to disturb the bottle after it has been inoculated. The photos show: uninoculated medium [1]; growth of strict aerobe (which also has produced a yellow pigment) [2]; growth of a facultative organism which prefers an aerobic environment [3]; growth of a facultative organism with less preference for aerobic conditions (movement away from the stab line may indicate organism is motile) [4]; growth of strict anaerobe (with no growth near surface) [5]. Thioglycollate medium is used in the sterility testing of sterile products. It contains a redox potential indicator dye which is coloured at the top of the medium [1], because it is oxidised due to the head space of air in the bottle. It is colourless in the rest of the medium because it is reduced. If more than the top third of the medium is coloured, it is too oxidised and not suitable for growing anaerobes and should be heated in a steamer to drive off dissolved oxygen before being used.

7 - SEMI-SOLID AGAR STAB This test is a less exact alternative to the hanging-drop test for motility. The organism under test is stab-inoculated into a tube of semi-solid nutrient agar and incubated at slightly below its optimum growth temperature (to increase motility). After incubation, motile organisms will have grown throughout the medium [1], while the growth of nonmotile organisms will be restricted to the stab line [2].

8 - BLOOD AGAR PLATE The ability of some bacteria to break down blood is a useful identification feature and a 'rough-and-ready' indication of pathogenicity. Some bacteria lyse blood cells completely and, when grown on blood agar, produce colonies surrounded by clear areas [1]. This is referred to as beta haemolysis (or total haemolysis). Other bacteria are able to partially break down blood cells. Such bacteria, when grown on blood agar produce colonies surrounded by greenish zones [2]. This is referred to as alpha haemolysis (or partial haemolysis). The inability to break down

blood cells is sometimes called gamma haemolysis (i.e. no haemolysis). Usually, blood agar plates are poured as a double layer - with a bottom half layer of plain, nutrient agar and a top thin layer of nutrient agar mixed with blood. The thin layer of blood agar is necessary to see any zones of haemolysis clearly. The genus Streptococcus contains many species which exhibit alpha or beta haemolysis and this feature is used in their identification.

9 - O/F TEST Hugh & Leifson's Oxidation/Fermentation test determines whether bacteria attack carbohydrates by oxidation or fermentation. The test is performed by stab inoculating the organism into two tubes of Hugh & Leifson's medium, with one of the tubes being sealed with liquid paraffin. If the organism is oxidative, there will be acid (yellow colour) in the aerobic tube only [1]. If the organism is fermentative, there will be acid in both tubes [2]. In some bacterial genera the test is not applicable, in which case neither tube will produce a yellow colour. Two points to note are: a) the tubes may need up to 14 days of incubation and b) not all organisms may grow in Hugh & Leifson's medium.

10 - ANTIBIOTIC SENSTIVITY Sometimes, an organism's sensitivity to certain antibiotics provides confirmatory evidence of its identity. More importantly, such tests are routinely performed by pathology laboratories on clinical specimens from patients to decide which antibiotic to prescribe to treat their infection. Such data also provide information about the patterns of antibiotic resistance being exhibited by pathogens at that particular time in that particular geographic location. This information enables doctors to use "best guess therapy" to prescribe antibiotics immediately, without waiting for sensitivity tests to be performed. Antibiotic sensitivity is tested by preparing a plate of nutrient agar which has been seeded with the test organism. After the surface has been overdried, a special filter paper template is placed on the agar surface. The projections on the template have been impregnated with a variety of antibiotics which diffuse out into the agar gel during a short holding period before the plates are incubated. Different templates impregnated with different antibiotics are used depending on whether the organism is Gram-positive [1] or Gram-negative [2]. After incubation for 24hr, zones of inhibition of growth can be seen round those projections impregnated with an antibiotic to which the organism is sensitive. Resistance to an antibiotic is shown by growth of the organism right up to the template.

11 - SEROLOGICAL TYPING Rebecca Lancefield devised a system for serotyping streptococci based on differences in a carbohydrate antigen in the streptococcal cell wall. Most (but not all) streptococci can be serotyped by her technique and are allocated a Lancefield's Group. The technique involves extracting the carbohydrate antigen in solution with a proteolytic enzyme at 37 C for a short period and then testing this antigenic extract solution against a number of different antisera containing antibody against the Lancefield's Group antigens. In the first photo below, a drop of antigenic extract from six different streptococci has been placed on the black circles. Each drop has then been mixed with a drop of Lancefield's Group D antiserum. Agglutination in circle number 3 (seen as flecking or curdling of the suspension) shows that organism No.3 belongs to Lancefield's Group D. No agglutination is evident in the other circles. A close-up of a negative result (i.e. no agglutination; circle No 2) and a positive result (i.e.agglutination; circle No 3) is shown in the bottom photo below. Lancefield's Groups have some clinical significance: streptococcal throat and skin infections are usually caused by organisms in Lancefield's Group A (e.g. Streptococcus pyogenes), while streptococci inhabiting our gut belong to

Lancefield's Group D (e.g. Streptococcus faecalis - now called Enterococcus faecalis).

Check it out! You might expect to use a precipitin reaction in the Lancefield's Grouping technique because you made a soluble antigenic extract with the proteolytic enzyme. Why, in fact, is it demonstrated by an agglutination reaction? Work out the answer and click here to check whether you are correct.

The Streptococci