ZoBell — 52 — Marine Microbiology 



Lloyd (1930) that marine bacteria are not free-floating but usually occur 

 attached to solid particles. According to Waksman et al. (1933c), most 

 marine bacteria are found attached to larger plankton organisms, existing 

 "only to a very limited extent in the free water of the sea." Similarly, 

 most of the bacteria in mud occur attached to or adsorbed on solid par- 

 ticles. When plated, each particle, mass, clump, or group of bacteria will 

 give rise to only one colony, whereas many individual cells associated 

 with such particles or groups may be distinguished microscopically. 

 However, direct counts are attended by many technical difficulties and 

 have limitations which restrict their usefulness. At best, direct counts 

 give data which only supplement and aid in the interpretation of results 

 obtained by cultural procedures. 



If the material to be examined contains more than a million bacteria 

 per ml., a modified Breed and Brew (1916) method can be employed 

 for their enumeration. After treating the material with 0.02 per cent 

 gelatin to serve as a fixative, o.oi ml. is transferred to a clean glass slide 

 with a capillary pipette or a platinum loop calibrated to deliver o.oi ml. 

 This material is spread evenly over exactly i.o sq. cm. of the glass slide by 

 using guide plates designed for this purpose or by marking off a square 

 centimeter with a grease pencil. After drying and staining, the smear is 

 examined microscopically using the oil immersion objective of a compound 

 microscope equipped with a mechanical stage. If the field of vision of 

 such a microscope is 1/5000 sq. cm., the average number of bacteria seen 

 in a microscope field multiplied by 500,000 represents the number of bac- 

 teria per ml. of the original material. The area of the field of vision of 

 each microscope must be determined from the diameter of the field. 



Either carbol-erythrosin or rose bengal consisting of i.o per cent of 

 the dye and 0.02 per cent anhydrous calcium chloride in 5 per cent aque- 

 ous phenol solution, as recommended by Conn (1918) for the direct 

 microscopic study of soil microorganisms, has given good results for stain- 

 ing smears of marine microorganisms. 



Tarr (1941) has described a modification of the Breed and Brew 

 direct microscopic method for counting bacteria in fish flesh. 



Employing a shallow haemocytometer resembling the Petroff-Hausser 

 counter described below and a dark-field microscope, Amann (191 i) found 

 nearly 80,000 bacteria per ml. of spring water and only 600 by plating 

 procedures. However, i\MANN emphasized that accurate or reproducible 

 counts require the examination of numerous samples. 



After investigating various direct and cultural methods for enumer- 

 ating bacteria, Wilson and Kullmann (1931) stated that "The direct 

 count with the Petroff-Hausser or similar bacteria counting chambers 

 proved to be the most accurate of all the methods investigated and its use 

 whenever possible is advised." The Petroff-Hausser counter is a modified 

 haemocytometer having a chamber only 0.02 mm. deep which minimizes 

 the necessity of extensive focussing in order to locate bacteria in different 

 levels. The bacteria can be stained for light-field observation or they can 

 be observed unstained with the dark-field microscope. The disadvantage 

 of this method is that each bacterium per square on the Petroff-Hausser 

 counter represents 20,000,000 organisms per ml. of sample. Therefore 

 it can be used to advantage only when numbers of organisms are high. 



The concentration of organisms for direct counts : — If the material 

 to be examined contains fewer than a million bacteria per ml. (sea water 



