Chapter IV — 55 — Methods of Enumeration 



Direct counts on bacteria in mud : — Bacteria are usually sufficiently 

 abundant in marine bottom deposits so that it is not necessary to concen- 

 trate them preparatory to estimating their abundance by the direct 

 microscopic method, although there are other difficulties. Conn (1918), 

 WiNOGRADSKY (1928), CiiOLODNY (1930), Rossi (1936), and othcrs have 

 described methods for the direct microscopic study of soil microoiganisms. 

 Some of these methods are applicable to the enumeration of microorgan- 

 isms in marine sediments. 



Conn's (1918) method calls for mixing one part of soil with ten times 

 its weight of 0.015 P^^ cent gelatin solution. Exactly o.oi ml. of this is 

 transferred with a calibrated capillary pipette or platinum loop to a clean 

 glass slide and smeared evenly over exactly i.o sq. cm. The smear is air 

 dried, stained with carbol-erythrosin or rose bengal, and subsequently the 

 bacteria which appear in each of 10 to 100 fields are counted, using the oil 

 immersion lens. The average number of bacteria per field, multiplied by 

 a factor which must be determined from the area of the microscope field, 

 represents the number of bacteria per gram of soil sample. 



WiNOGRADSKY (1928) recommended centrifuging the diluted soil sam- 

 ple and the Examination of o.oi ml. of the resulting sediment as well as 

 0.01 ml. of the supernatant fluid. This gives additional information on 

 the spatial distribution of the bacteria in the soil. When applied to 

 marine sediments it is found that virtually all of the bacteria occur inti- 

 mately associated with particulate matter. The adsorption of marine 

 bacteria by particulate materials has been discussed by W^aksman and 

 Vartiovaara (1938) and by ZoBell (19436). 



Cholodny (1930) recommended the insertion of sterile glass slides 

 directly in the soil in slits prepared with a knife or spatula. The slides are 

 left there for one to three weeks during which time many bacteria become 

 attached. Then the slides are carefully removed by excavation. After 

 washing free of adherent dirt and fixing over a flame, each slide is stained 

 with erythrosin, carbol-fuchsin, or rose bengal. Obviously this method 

 can be apphed to studies of marine bacteria only in mud flats and in the 

 intertidal zone, but there it has given much information on the ecology 

 and morphology of microorganisms. Often Cholodny slides bear micro- 

 organisms which never appear in cultural material. The number of 

 microorganisms which attach suggests that a large proportion of bottom- 

 dwelling microorganisms are sessile, stereotropic, or thigmotactic. IMicro- 

 colony development gives some idea concerning the rate of growth of 

 bottom-dwelling microorganisms in situ. 



Rossi (1936) prepared impression smears by pressing sterile slides 

 against a freshly prepared soil surface and examining them at once after 

 fixing and staining. This method has contributed information on the 

 spatial relationships of microorganisms in mud flats studied by ZoBell 

 and Feltham (1942). It is also useful for investigating the distribution 

 of microorganisms in mud cores by making impression smears against 

 freshly cut surfaces of the cores. Such impression smears can be prepared 

 in the field for subsequent examination in the shore laboratory. 



Soil microorganisms are counted by the ratio method of Thornton 

 and Gray (1934) by diluting 5 gm. of soil with 25 ml. of indigotin suspen- 

 sion which contains about 200 million indigotin particles per ml. This 

 mixture of soil and indigotin suspension is diluted with 25 ml. of sterile 

 O.OI per cent agar solution and shaken for three minutes. A drop smeared 

 on a glass slide is permitted to dry, after which it may be stained with 



