Lesson 7 - Microorganism Detection and Control SAQ

** Describe a media and the principles used when selecting for each of the following microorganisms: 

a) Lactic acid bacteria (LAB)

b) Enterobacteriaceae

c) Pectinatus

d) Zymomonas

e) Brettanomyces

f) Non-Saccharomyces wild yeast

Response:

a) We can detect LAB using a number of media, including MRS, Raka-Ray, and NBB. We will discuss Raka-Ray, which is a media recommended by the ASBC and EBC. This media is a development of universal beer agar (UBA), in which stimulants are added to encourage growth and visualisation on plates. In addition to glucose, fructose and maltose are added as carbon sources for those Pediococcus and Lactobacillus species unable to utilise glucose. Gram-negative bacteria and brewing yeast growth are restricted by addition of 2-phenylethanol and cycloheximide. 

Incubation is anaerobic, lasting 4-7 days at 25-30°C on solid or semi-solid media. Longer times are needed for species that grow more slowly.

b) Enterobacteriaceae may be detected on MacConkey media. Lactose is used as a selective carbon source, and bile salts are added to inhibit Gram-positive bacteria. Neutral red dye is added to detect growth of acid producers.

We can use broth to select for acid forming gas producers, which are then grown on agar plates. After 24 hours at 35°C, growing colonies will be stained pink from the neutral red reaction. The media surrounding the colonies may be hazy from bile salt precipitation.

c) The Gram-negative anaerobe Pectinatus can be detected on a SMMP medium, which contains lactate as the carbon source. Reducing agents are added to promote the growth of anaerobic bacteria and cycloheximide, which prevents yeast growth. Gram-positive bacteria are inhibited with crystal violet and sodium fusidate. The liquid media is incubated at 28‐30°C for 14 days under anaerobic conditions and examined for turbidity, which indicates growth.

d) Zymomonas spp. are detected by Dadds and Martin media containing glucose, yeast extract, peptone supplemented with ethanol (3% v/v), and cycloheximide at 50 mg/L. They are then adjusted to pH 4. The resulting metabolic products, acetaldehyde and hydrogen sulphide, can be detected by colour formation using Schiff’s reagent (purple) and lead acetate (grey/black).

e) Brettanomyces will grow slowly on media where cycloheximide has been added to suppress brewing yeast. Malt extract liquid media supports growth after 5 days at 25°C, after which time the cells are examined. Colony formation is observed after six weeks growth on malt agar + 2% calcium carbonate.

f) Non-Saccharomyces wild yeast can use lysine as a sole nitrogen source,  whereas brewing yeast cannot. A solution containing 10⁷ brewing yeast is streaked onto the agar plate and incubated at 25°C. Growth is counted daily and expressed per 10⁶ yeast cells.