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Van der Wath, J.G.

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Owletsiezwmt Jow-rwl o.f Veterinary Science and Animal Industry, Folume 23, N11>rnhe1·s 1 and 2, ,~!a1'clt , 1948. IPrinted in the Union of South Africa by the Gove rnm ent l'ri11te r, Pretoria. Studies on the Alimentary Tract of the Merino Sheep in South Africa. XII.-A Technique for the counting of Ruminal Bacteria. By J. G. YAK DEl{ \VA 'I'll , Rertion of Pln·siolog~·, OJ}(len>tepomt. IK the course of resean·he:-; on the digec;ti.Ye pnweRses in the nnnen of sheep it was realised tha t a n•liable methocl of estimatin g the bact erial populatiou in the rumina] ma ss \\·ould be inYaluable. In attempti ng to eYolve such u technique several <lifficulties hacl to be over('ome. Firstly, the method to he employed, secondly the moRt effective fixative a]l(l stain, and thirdly, the best t ime to make the nnmts so a :-; to obtain nw;;t uniform r esults. \Vith regard to the la stuamecl poi11t, it was found best to withdnnv rnminal ingPsta approximately 12 hours niter colllplet.ion of feerl in g . It was realiRed at th e outset that the plat ing all(l vVright's methods couid not be emplo:·P<l. In plating rumen ('Ontenh; t h e m edium usually becom es so overgrown by various hade1·ia HJL(l fungi, that it becomes impor;-sible to distinguish coloni e;, . Selel·tive media were abo trie<l lmt without RtH.:eess. According to L eishmnnH (lDlO ) the re~uli ~ 11·it lt \Vright's metho<l are not c-onsisten t anll !:'nm·s of 50-100 per ceut . nwY OlT\U. 'l'h e enumeration of bacteria (for t he purpo:->efl of ,;tauclanlising- h:lCterial emulsion ,; for vaccines) in a haemocytomet!:'r cl1amh<'r, \\·ar; sugg-e:-;te<l by Mallmv an<l Homer Wright (1908). They employee] a well 0·02 nnn. <leep, an optically plaue coverslip , a 1 / 16 inch dry lem.; and 110 staining fluid. In 1908 Leith Murray likewise used an ordinary 'l'homa-7;eiss haemocy tometer with a well 0 ·1 mm. deep, an ordi~ary covPrslip, a 1 / 12 oil immersion lens and n stai11ing solution of weak G.1emsa. TECHNIQUE. For the purposes of thi r; work, the <IUthor F>eledecl a Petroff-Hauser bacteria l counting chamber , whieh has a well 0 · 02 mm. deep . This cham her has a r einforced preeiRiou C{JVeJ·slip, optically phme and 0· 18 mm. thick. The apparatus can be use<l with <lark ancl bright field illumination and is suitable for all achromatic· oil immer sion len ses as well as dry lenses. Fnrther advan tages are (Gl~·nn, Powell et al, ]!)1 3) that almost all the bacteria settle to the bottom of the c-hamber in five to ten minutes, whe11 acc-urate counts can he mad e. BadPria adhering to the uuder surface ·of the coverslip , and any still floating· in th e cl1am her, are easi l~- enumerated. The optical definition of tl1 e bacteria is go()(l owing to the small quantity of fiuid. 385 STUDIES OK THE .\Lili[J.:~·T.-\RY TRACT OF ME111NO ::>HEEl'. XII. After trying out several stains and methods of killing the bacteria suspended in the ruminal fiuid, a five per cent. <;arbolic solution or 30 per cent. hydrogen peroxide was found to be most suitable, ~~lthoug-h not entirely effedive, probably due to the great variety of org-musm;; present. Fresh rumen ingesta has a definite clecolorising adion on stains, and unless the concentration is inc1·eu sed several times beyond that required for ordinary bacterial emulsions, the stain is redueed to sueh an extent that it loses its staining power almost completel~r . A further point that had to be <·onsiuered was the f<1ct tlwt staining is most effective when a stain is so used that its hydrogen-ion eoneentration is ehanged as little as possible, so that the stain is allowed to act at the pH where it stains be,;t. 'fhu,; a sta in which would be effeetive at a pH of approximately 6·8 to 7·2 ha(l to be ,;ought. For this purpose nile blue sulphate in a one per <:ent . <·on cen-trntion was found to be most suitable. H a strong·er f'Oll('entration is use<l the organi;;ms are incline(] to clump . Afte1· couside1·able experimentation the following routine method was adopted in preparing material for counting:-Ruminal ingesta i;; with(lrawn into a sterilised glass tube and ehilleu for fifteen to thirty minutes in iee cold water, ~>o as to stop fermentation and break the froth. After this the tube is shaken for a few secouclf; to mix the material and one eubi(: centimeter of it is drawn into a wide-mouthed 2 r.c. pipette. 'rhis is run into a small a c.c. gla~;~; tube with storlper. To this 0 . 25 C.('. of a 5 per <·ent. earbolic- tH'i(l solutiou, or 30 p!:'r <·ent . l1Yrlrogen peroxide i~; a<lcled. The mixture is ~;tined with a t h in glas,; rod and then allowed to stand for one hour. After this 0·3 c.c. of nile blue sulphate is added an<l the mixture wE'll st irred. After staining for a11 hour 1 C . (·. -of thE' ~;tn ined material i~; pipette<l off and mixed with 257 c.c. of sterili sed distillf'cl water, the pipette being \Yashe<l out ''"ell in this watf'r. In this way a dilution of 1 in 400 is obtained whi<:h was found to he the most imitable dilution for ordinary counts. vVb en bacterial populations were low the dilution wa s reduced to 1 in 200. Unstained barteria can he counted in a similar waY, but it waR fouJl(l that by staining, f'yestrain was mu('h reduced an(l -accuracy inerease<l . After the final dilution is made the suspension is allowe<l to st:md for two minute,· RO as to allow the ('Oarser material to settle . \Yith a Thoma-Zei~;s pipette a clrop of the emuhion is allowPd to pa~;R un<lPr the coverslip b~- eapillmT attraction. 'flw c-ounting chamber i~; then plac-ed in po;;ition on a level microscovic Rbltte arHl allowed to stancl for three to five minutes in ·order to allow the- bacteria to settle. 'ren blocks of nine small squares are then counted, giving- the total number of baeteria in 90 squares. 'l'be Yolume of ea<·h square being· 0·0008 <:uhic mn1., the total volunw is 0 -072 eubi(' mm. If N = totalnumlwr of organisms in 0·072 cuhi(· mm., then with a dilution of ] in 400 there would he 400N org-aniRms in 0·072 cuhi(' mm . 'rhus in on e cubi(· mm . there 'nnrld be 4~~~ organism fi . If the number of bacteria in 90 small squares is known , the total number per cubi c mm. coul(l thus lw ('al<-ulatPd b:- multiplying- "-ith .~07°2 for a dilution ·of l in 400 or -~~~ for a (lilution of 1 iu 200 ancl so forth. These counts are of course not necessarily hue total rounts, smce an unknown percentage of organiRmR penetrate into or become adsmbed to food particles. The total eount as given must, therefore, be considered always 386 J. G. VANDER WATH. as lwing- less than the true com1t would he. In exam ining food particles and ruminnl fluid micrmwopirally after :fixation and staining for bacteria, it is ~een that onlY n very small proportion of the orga11isms is not counted, <lue to illtimate relation;,;hip with food partides. Throughout the investigatio11s Zeiss Hu:v·g·ens oculars 7 x and a Zeisr; A<-hrmuate 40 x dry lPll R WPI'e n se1l with ex<-ellellt reRults. REFERENCES. LEISHMANN (1910). MALLORY AND HOMER WRIGHT LEITH MURRAY (1908). (1908). REES AND COX (1913/ 14). 'l'he Jnl. of l For these references see GLYNN, POWELL, Path and Bact., Vol. 18, pp. 379- 400. nnd/ or LEIBHMANN (1901). Jnl. Roy. lnst. Pub.l Health, Vol. 18 (8), p.46l. KNAYSI, G. (1925). MALLORY AND WRIGHT (1908). Pathological)' 'l'echnique, London. MURRAY, LEITH (1908). Lancet, Vol. 1, p. 7!10. 387 No.2. Jnl. of Bact. . Vol. 20. 

Studies on the alimentary tract of the Merino sheep in South Africa. XII. A technique for the counting of ruminal bacteria

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Studies on the alimentary tract of the Merino sheep in South Africa. XII. A technique for the counting of ruminal bacteria

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