LIEU AUIOTNAD A NU MICROS: }OPIC OBSERVATION OF SIMPLE STAINED BACTERIA A 0 rere Simple staining is a technique by which cells are stained by one dye. tained cells are visible because of the increased contrast between the cells and the background. Once the bacteriaare stained, their size, shape and cell arrangement could be determined. However success in bacterial staining depends first of all on the preparation of thin smear of the organisms. HL ossecrives . To prepare bacterial smears from culture materials. 2. To stain the smear with a single stain such as methylene blue, basic fuchsin or crystal violet. 3. To show how microorganisms are so visualized and to determine their size, shape and cell arrangement. _ Broth cultures Of Bacillus megaterium , B. coli , Proteus spp. and , Spee aureus. . Yeast and E. coli cultures on YPG. . Slides . Inoculating loop Dye . Staining rack. . Bunsen burner. QQXU AY DSnum Loop t Ce BO AO LEE A Lal ) Remove Gatton plug fatto falta wit cand (fig.b), SAA ©) Pass (flame) mouth of the broth cull flame.(fig.1.b), Teed d) Insert the sterile inoculating loop in to the cult loopful of bacterial culture to the center of ck (fig.1.c), ©) Spread the microorg: loop. (fig.1f). sad * ichiare anism over a wide area of the slide i 9 2 ) Plame-sterilize the loop & lay aside (fig.1.g). is 8) Heat fix the smear on the slide by passing the slide 3 times or the top of the Bunsen bu ner. Be sure that the film is fac fixing the smear coagulates some of th, ing then to the slide, fe cell proteins making h) Allow the slide to cool on staining rack. i . From an agar cultures: a) Place a small drop of water on the slide, moculating loop, 3 ©) With sterile loop pick up a ve sterile ty small amount of b: .cterial colony a emulsify itin the drop of water A b) Flame- sterilize the end of the i diameter, ©) Heat-fix the smear, B. Technique of the simple 1. Cover the bacteria! Stain, smear with dye like methylene blue for 1-2 mins., ith water to Temove the ©Xcess stain and blotd 3, Examine the slide under the my i ieroscope g of bacteria and yeast aud 2. Rinse the slide wivaqm oun Jo yoau oy} ouey pue JOU Par sauurooaq I Snjd aqn ayy erowoy 'q | [HUN doo] oy) cure] e ~S gL OCINE TAMICROSCOPIC OBSERVATIONS OF 1 MICROORGANISMS IN THE LIVING STATE a Unstained organisms are difficult to ‘observe by normal microscopy since their refractive index is only slightly different from that of the suspending medium. However, certain characteristics such as motility can be demonstrated by wet mount preperation of bacterial culture. Motility must be distinguished from Brownian movement which is vibration of cells in a solution. Most-motile bacteria often show the Brownian motion, while motile bacteria move over long distances in a definite direction. aE To view various types of microorganisms in living state & determine it’s shape, size & motility. 1. Nutrient broth cultures of Bacillus megaterium, E. coli - Proteus SPP» Klebsiella spp., Staphylococcus spp., Spirillum & yeast. . Cyanobacteria - Nostoc & Gleocapsa 2 3. Pasteur pipets | 1, Wash a slide with soap and water. 2. Dry the slide and pass it 3-5 times through the B - grease and oil. junsen flame to remov' 3. With sterile pasteur pipet, place one drop of 4 ; p of the bacteri: center of a cleaned slide and cover with a cleaned ee a er slip. 4, Examine the bacteria and cyanobacteria und ler reduei the high power dry lens (oil-immersion must not Nee ly 5, Record your observation in a table and sketch th : e microorganism. é 14