CHAPTEE VI. 



MICROSCOPIC METHODS. 



THE PREPARATION, STAINING, AND MICROSCOPIC EXAMINATION 



OF BACTERIA. 



THE direct microscopic examination of suspected substances for 

 bacteria can be made either with or without staining. Unstained, 

 the bacteria are examined in a hanging drop or on transparent media, 

 under daylight, or, better, artificial light to note their motility, their size 

 and form, and their general arrangement; but for more exact study 

 of their appearance they can be so much better observed when 

 stained that this step is always advisable. 



Elimination of Foreign Bacteria from Preparations. Since bacteria 

 are present in the air, in dust, in tap water, on our bodies, clothes, and 

 all surrounding objects, it follows that when we begin to examine sub- 

 stances for bacteria the first requisite is that the materials we use, 

 such as staining fluid, cover-glasses, etc., should be practically free 

 from bacteria, both living and dead, otherwise we may not be able to 

 tell whether those we detect belonged originally in the substances 

 examined or only in the materials we have used in the investigation. 



Film Preparation. A cover-glass or slide preparation is made as 

 follows: A very small amount of the blood, pus, discharges from 

 mucous membranes, cultures from fluid media, or other material to 

 be examined is removed by means of a sterile swab or platinum loop 

 and smeared undiluted in an even, thin film over a perfectly clean, 1 

 thin cover-glass or slide. From cultures on solid media, however, 

 on account of the abundance of bacteria in the material, a little of the 

 growth is diluted by adding it to a tiny drop of clean distilled water 

 which has been previously placed on the glass. The amount of dilution 

 is learned after a few trials. It is best to add to the drop just enough 

 of the culture to make a perceptible cloudiness. The mixture is then 

 smeared thinly and uniformly over the glass. When blood or pus is 

 to be studied it is well to put a small drop on a slide or cover-glass 

 and then immediately to place on top of this another. The fluid will 

 spread between the two, and when they are drawn apart a fairly even 



1 To render new cover-slips clean and free from grease, place them in strong nitric acid for a few 

 hours, then rinse them in water, then in alcohol, then in ether. Place them finally for keeping 

 in alcohol, to which a little ammonia has been added. When used wipe with soft, clean linen or 

 cotton cloth. If old cover-slips are used, boil first in soda solution. Another procedure is to soak 

 the cover-glasses first in alcohol, then wipe with soft linen, then place in a Petri dish, and heat in 

 the dry sterilizer for one hour at 200 C. A cover-glass is not clean when a drop of water spread over 

 it does not remain even, but gathers in droplets. 



