MILK FERMENTERS David B. Fankhauser, PhD Professor of Biology and Chemistry U.C. Clermont College Batavia OH 45103 9 July 1995, rvsd 28 June '96, 7 July '97, 23 Feb. '99, 18 Apr 99, 12 July 00
Gram stain of yogurt, 1000x		Gram stain of yogurt, 1000x

Milk is extremely perishable and many means have been developed to preserve it. The earliest one which has been used for many thousands of years is fermentation. Milk can be fermented by inoculating fresh milk with the appropriate bacteria and keeping it at a temperature which favors bacterial growth.  As the bacteria grow, they convert the sugar in milk (lactose) to lactic acid.  You can detect its presence by the sour taste (sour is how we taste acid).   Lactic acid preserves the milk by lowering the pH which prevents the growth of putrefactive and/or pathogenic bacteria who do not grow well in acid conditions.

Fermentation is a means by which cells growing anaerobically dispose of excess hydrogen atoms generated during the breakdown of sugar, known as glycolysis.  It is defined biochemically as the catabolism of glucose (or other sugars) in which the terminal hydrogen acceptor is an organic molecule (carbon containing).  In lactic acid bacteria, they "dump" excess hydrogens on to pyruvic acid, the breakdown product of glucose.  This produces lactic acid.  Our muscles do the same thing, which causes the sting in over exercised muscles.  In all fermentation, a hydrogen carrier (NAD) is freed up to assist further with glycolysis.  Yeast too performs fermentation, but with different terminal hydrogen acceptors (acetaldehyde) and products (CO2 and ethanol).  You will note that alcoholic fermentation is also an anaerobic process. Since the terminal hydrogen acceptor in each of these microbiological processes is an organic molecule, they are, by definition, fermentation.

In contrast, respiration uses an inorganic terminal hydrogen acceptor (such as oxygen). If oxygen is the acceptor, then water is produced.

Casein, the predominant protein in milk, is soluble at a neutral pH, but insoluble in acid.  Thus when milk sours, casein precipitates and which thickens the product. Numerous strains of bacteria are capable of converting lactose to lactic acid.  We will look at several fermented milk products to study their morphology and staining characteristics.

1. Make a thin smear of each milk product well spaced on the same slide, labeling with a wax pencil Y, B and S. (see protocol Smear and Staining of Bacterial Specimens)
2. Stain them according to the procedure for the Gram stain (see related protocol Gram Stain Protocol), or any simple stain such as methylene blue, should you only be interested in seeing bacterial morphology.
3. View the stained smear at 400x to determine the characteristic features, select a field which is well spread and typically stained. Then switch to 1000x with oil. (The oil immersion lens is challenging to novices.  Do not use this lens unless you have been instsructed in its use.)
4. Illustrate typical fields for each milk product showing all observed morphologies of bacteria. Label the morphologies and their probable identities according to the following type of bacteria expected in these fermented milk products:

BUTTERMILK  is the fermentation of milk by a culture lactic acid-producing Streptococcus lactis  plus Leuconostoc citrovorum which converts lactic acid to aldehydes and ketones which gives it its flavor and aroma.

SOUR CREAM  is produced by the same bacteria as buttermilk, but the starting milk product is pasteurized light cream. Bacteria are less numerous than in buttermilk.