Research-Article| Volume 21, ISSUE 3, P129-141, March 1938

A Method of Counting Viable Bacteria in Milk by Means of the Microscope1

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      A direct microscopic method for distinguishing dead from viable bacterial cells in milk is reported. The method is based on the principles of vital staining. One cc. of the milk to be examined is mixed with 0.5 cc. of a dye mixture containing 0.1 per cent methylene blue and 0.1 per cent Nile blue sulfate, and with 0.5 cc. of a gelatin-agar mixture containing 8 per cent gelatin and 2 per cent agar. 0.01 cc. of this mixture is placed on a clean glass slide and covered with a small cover glass (1.28 × 1.28 cm.). The stained cells are then counted and they represent the dead cells already present in the milk. To a similar mixture one drop of 0.7 to 0.8 normal NaOH is added. This causes staining of all the cells present in the milk. The difference between the two counts represents the number of viable cells present.
      The object of the agar-gelatin mixture is to solidify the preparation, keeping the bacteria in position. The factor is calculated from a knowledge of the amount of milk present in the preparation and the area of the cover glass.
      When desirable, fat can be removed by shaking the milk with heptane followed by moderate centrifuging.
      A number of tables are included showing the results obtained when the method is applied to pure cultures and to pasteurized milk. The counts obtained are also compared to petri plate counts made on the same material.
      The effect of pasteurization on the stainability of bacteria in milk is studied and briefly discussed.


        • Beattie M.
        Use of a differential stain in the direct enumeration of bacteria in pasteurized milk.
        Am. Jour. Pub. Health. 1927; 17: 1031-1034
        • Benians T.H.C.
        Belief staining for bacteria and spirochaetes.
        Brit. Med. Jour. 1916; 2: 722-723
        • Drobot’ko V.G.
        Okraska mikrobov v “mertvyitavet” kak reaktiv na sostoianie mikrobnoi abolochki.
        Mikrobiologiia. 1934; 3: 75-78
        • Hall H.C.
        Untersuchungen über die Bedeutung des Petroläthers fur den Nachweis von Typhus- und Paratyphusbakterien im Stuhl.
        Berl. Win. Wochenschr. 1915; 52: 1326-1330
        • Hastings E.G.
        • Davenport A.
        The effect of pasteurization on the number of bacteria in milk when this is determined by the direct microscopic count.
        Jour. Dairy Sci. 1920; 3: 494-501
        • Henrici A.T.
        Differential counting of living and dead cells of bacteria.
        Proc. Soc. Exp. Biol. Med. 1923; 20: 293-295
        • Kayser H.
        Die Unterseheidung von lebenden und toten Bakterien durch die Farbung.
        Centralbl. f. Bakt. I, Orig. 1912; 62: 174-176
        • Knaysi G.
        A microscopic method of distinguishing dead from living bacterial cells.
        Jour. Bact. 1935; 30: 193-206
      1. Knaysi, G., and Naghski, J. Unpublished. 1936.

      2. Krukovski, V., and Sharp, P. F. Unpublished. 1936.

        • Luyet B.J.
        Differential staining for living and dead cells.
        Science. 1937; 85: 106-107
        • Morel A.
        • Rochaix A.
        • Mathais C.
        Recherches sur les pouvoirs infertilisant et antiseptique de quelques hydrocarbures vis-à-vis des cultures de certains microbes pathogènes.
        Comp. Rend. Soc. Biol. 1931; 108: 897-899
        • Nadelin E.P.
        Methods for differential staining for dead and living bacteria.
        Milk Dealer. 1937; 26: 35
        • Proca G.
        Sur une coloration différentielle des bactéries mortes.
        Comp. Rend. Soc. Biol. 1909; 61: 148-149
        • Růžička V.
        Über tinetorielle Differenzen Zwischen lebendem und abgestorbenem Protoplasma.
        Pflüger's Arch. f. d. ges Physiol. d. Mensch. u. d. Thiere. 1905; 107: 497-534
        • Sherman J.M.
        • Cameron G.M.
        Lethal environmental factors within the natural range of growth.
        Jour. Bact. 1934; 27: 341-348
        • Walbum L.E.
        Undersøgelser over Petroleumsaethers og nogle rene Kulbrinters Indirkning paa Tyfus-Coligruppens Bakterier.
        Det. Kgl. Danske Vid. Selsk. Biol. Medd. 1918; 1 (Nr 5): 1-48
        • Ward A.B.
        • Myers C.E.
        Influence of dead bacteria on microscopic counts of pasteurized milk.
        Am. Jour. Pub. Health. 1937; 27: 899-905