Lab class 3 - Detection of specified organisms and identification of bacteria

These topics are covered together so you will, during this class, interpret tests to detect the presence of specific

objectionable organisms and the results of a testing scheme designed to identify and distinguish intestinal

bacteria. You will have to complete an information table in the lab book before coming to the class and find

information about bacterial detection and identification methods. The assessment will be based upon the

correctness and completeness of ALL the tables in the schedule.

Before coming to the class complete Table 1 in the lab schedule and find out the following:

1. To which categories of pharmaceutical products are the various tests for specified organisms applied?

Tests for the detection of four named bacteria (Escherichia coli, salmonellae, Pseudomonas

aeruginosa and Staphylococcus aureus) in pharmaceutical raw materials and finished products are described
in the United States Pharmacopoeia (USP) (1995), and in the European Pharmacopoeia (EP) (1997) and British
Pharmacopoeia (BP) (1999) (where the tests are identical). The EP and BP additionally describe a test for the
detection of Clostridium perfringens. These organisms are singled out for special attention in this way
because they represent particular infection hazards to the patient, or because their presence is a criterion
of the quality of raw materials.
The detection tests are typically applied to raw materials of natural or biological origin (starches, gums,
gelatin, talc, etc.) where there may be a background of unrelated organisms which may grossly
outnumber the species of interest.

2. The principle of Gram staining, i.e. what reagents are added and in which order, what colours

represent positive and negative Gram stain results

Gram Stain:

1. Prepare a thin smear on a slide and heat fix

2. Add crystal violet and leave for 30 seconds

3. Water rinse for 2 minutes

4. Wash off the water with Lugol’s iodine and leave for 30 seconds

5. Differentiate with 95% ethanol

6. Water rinse

7. Counterstain with safranin for 2 minutes

8. Water rinse and blot

Gram negative – pink ; Gram positive – purple.

3. The Gram stain reaction for the organisms listed in Table 1 see question 5

4. What represents a positive result in each of the tests in Table 1? Staph. aureus – positive coagulase test;

E. coli – Indole production at 44 0C; Salmonellae – TSI yellow butt, pink slant ; P. aeruginosa – positive

oxidase test.
5. Which of the organisms for which the pharmacopoeias describe detection tests are Gram-positive and

which Gram-negative? Staph. aureus – gram positive (purple gram stain); E. coli, P. aeruginosa and

Salmonellae – gram negative (pink gram stain)

6. Which are the common gut bacteria and which common media are used to distinguish them?

MacConkey Agar, deoxycholate citrate agar, XLD agar, Brilliant green agar and TSI slants.

Escherichia, Klebsiella, Salmonellae and Enterobacter spp.

7. What indicators are used in the media recommended for E. coli and salmonellae and what colour

changes do they exhibit in acid and alkali? MacConkey agar – neutral red (acid-red colonies, alkali-

orange/brown colonies) E.coli will give red colonies + surrounding agar will be red. XLD & TSI slants

– phenol red (acid-yellow, alkali-pink/red). Salmonellae give red colonies with or without black

centres in XLD and yellow butt with pink slope in TSI slants.

8. What do XLD medium and triple sugar iron agar contain, and what reactions may be exhibited by the

various bacteria that may grow in them?

XLD – yeast extract, L-lysine, xylose, lactose, sucrose, sodium deoxycholate, sodium chloride, sodium

thiosulphate, ferric ammonium citrate, phenol red and agar

TSI – agar, phenol red, lactose, sucrose, glucose and dextrose, sodium thiosulphate and ferrous

sulphate or ferrous ammonium sulphate.

Reactions:

- Sugar fermentation ( lactose, sucrose, glucose, xylose, dextrose)

- Decarboxylation of L-lysine (XLD)

- Reduction of thiosulphate to give H2S

- H2S reacting with ferrous sulphate / ferric ammonium citrate to give black precipitate

- H2S production is retarded in alkaline conditions (i.e. black precipitate may not be present)

- Gas production, CO2 from sugar fermentation (TSI)

- Oxidative reactions – organisms that cannot ferment any sugars eg. P. aeruginosa oxidatively

decarboxylate peptides to give amines at the agar surface and these alkaline products give a pink

colour to the medium (TSI).

9. Of the bacteria in the following categories, which are lactose fermenters: (a) the common gut bacteria;

Escherichia, Klebsiella, Citrobacter and Enterobacter spp (b) bacteria that are the subject of

pharmacopoeial detection tests? E. coli

10. What distinguishes E. coli from some Proteus species that are also indole positive? Only E. coli

produces indole at 440C.

11. What is the principal feature distinguishing E. coli and salmonellae?

XLD

- Salmonellae can ferment the sugar xylose to produce acid. After exhausting xylose supply,

Salmonellae colonies will decarboxylate lysine, increasing the pH once again to alkaline. Red

colonies with/without black centres, agar will be red.

- E. coli will ferment lactose and sucrose to an extent that will prevent pH reversion by

decarboxylation and acidify the medium turning it yellow.

- E.coli ferments lactose, Salmonellae do not.

12. What are the principal components of Kovac's reagent and oxidase reagent?

Kovac’s reagent – isoamyl alcohol, p-Dimethylaminobenzaldehyde, concentrated HCl.

Oxidase reagent – N, N, N1, N1 – tetramethyl-p-phenylenediamine dihydrocholide suspended in

dimethyl sulphoxide or N, N dimethyl- p – phenylenediamine.

13. How specific are the various confirmatory tests: do they only give a positive result with one species or

with several? Not specific – give positive results for a number of bacteria.

14. What is the significance of the coagulase test and what does it indicate about pathogenic potential?

- Differentiates coagulase +ve bacteria from coagulase –ve bacteria.

- Differentiates Staph. aureus from Staph. epidermis.

- Coagulase reacts with prothrombin in blood, which enables protease enzyme to convert

fibrinogen to fibrin, resulting in clotting of blood. Coagulase is tightly bound to the surface of

Staph. aureus and can coat its surface with fibrin upon contact with blood. It is proposed that this

fibrin coating allows the bacteria to resist phagocytosis making the bacteria more virulent.