MICRO BIO LAB CHAP6.pdf

MICROBIOLOGY LAB LABORATORY 6: BACTERIAL STAINING Inoculating Loop & BACTERIAL STAINING Water Bottle The majority of microorganisms are studied in stained preparations. Unstained bacteria are visible, but the...

MICROBIOLOGY LAB LABORATORY 6: BACTERIAL STAINING Inoculating Loop & BACTERIAL STAINING Water Bottle The majority of microorganisms are studied in stained preparations. Unstained bacteria are visible, but the shapes, sizes, and appendages are Reagent: Crystal difficult to observe. Violet 3 Major Methods of Staining: 1. Simple 2. Differential 3. Special MATERIALS Reagent: Gram’s Iodine Bacterial Cultures (E. coli, S. aureus, B. subtilis, & unknown bacteria) Reagent: 95% Alcohol Lamp alcohol Glass microscope slides & Kim wipes Reagent: Safranin Reagent: Methylene blue MICROBIOLOGY LAB LABORATORY 6: BACTERIAL STAINING Basic or Positive Dyes ○ Types of dyes that are positively PROCEDURES charged. Contains cationic PREPARATION OF BACTERIAL SMEARS chromophores. The preparation of a good smear is the ○ Since all cells are negatively backbone of all the staining techniques charged, there is an attraction performed throughout this course. between the positive charge of A good smear is the result of the the dye and the negative charge mastery of 3 individual concepts: of the cell. 1. Heat fixing/adhering the cells to the Some common basic dyes used in slide so that they are not washed off staining are: during subsequent staining procedures. 2. Heat fixing gently so as not to cause ○ Methylene blue distortion of the cells. ○ Crystal violet 3. Preparing a thin smear, because the ○ Basic fuchsin thickness will determine whether you However, not all dyes are positively can visualize individual cells, their charged. arrangement or details regarding Gram Acidic or Negative Dyes reaction or internal structure. ○ Are negatively charged dyes, 4. Preparing a smear that isn’t too thin, so containing anionic organisms have been transferred to the chromophores, and are repelled slide. by the negatively charged bacterial cells. SIMPLE STAINING ○ They are usually used for Simple stain negative staining protocols The use of a single stain or dye to color which are the subject of a later a bacterium. laboratory. A single dye is used to color the Common acidic dyes are: otherwise colorless bacterial cells. ○ Nigrosis ○ India ink ○ Eosin MICROBIOLOGY LAB LABORATORY 6: BACTERIAL STAINING ❖ Bacteria are characterized based on the arrangement of cells: Diplo - cells arranged in pair Strepto - cells arranged in chains Staphylo - cells arranged in clusters Tetrad - cells arranged in four. ❖ Morphology and arrangement are Simple stain of Escherichia coli using the determined microscopically and must be basic/positive dye Methylene blue. identified in areas of your field of view ❖ Staining microbial cells is important where the cells are not crowded together because without stain, most bacterial because of a smear that is too thick. cells are extremely difficult to see. ❖ Overcrowding should not be confused ❖ Staining allows them to be seen so that with true staphylo- arrangements. observations as to their morphology These arrangements are the (i.e., individual cell shape) and result of bacterial reproduction arrangement (i.e., how the cells or binary fission. remain physically attached to one When fission of the cell is another after cell division) can be incomplete, due to the genetic made. programming of the cell, cells ❖ Bacteria are morphologically may remain physically attached categorized as either: to another, causing the bacterial Cocci - round arrangements. Bacilli - rod-shaped Tetrad and staphylo- Spirilla - spiral-shaped arrangements of cells result Spirochetes - corkscrew from binary fission along more than one plane of division and are only possible when cocci reproduce. You will never see bacilli or spirilla MICROBIOLOGY LAB LABORATORY 6: BACTERIAL STAINING arranged in tetrads or The positively charged stain will not clusters. bind to the cell wall; rather, it will form a deposit around the bacteria and produce a dark background so that the bacteria will appear as unstained cells with a clear area around them. GRAM STAINING Gram Stain Is a differential staining technique which uses the principle of timed, sequential applications of a primary stain, mordant, decolorizer, and finally a PRINCIPLES counterstain which differentiates To observe the overall bacterial between Gram-negative and morphology of a microorganism, harsh Gram-positive bacteria by virtue of staining or fixing techniques should be their difference in the cell wall avoided because these denature component. (coagulate) the protein found in ○ Mordant samples. The mordant is a ○ Denaturing alters the shape and substance used in size of cells. conjunction with a dye ○ Also, some cell types like to increase its staining spirochetes do not stain well, ability. For example, in even with common staining Gram stain, Gram's procedures. iodine is used to form a Negative Staining or Indirect or complex with crystal Background Staining violet that makes the ○ Is done by suspending bacteria dye molecules larger with a negatively charged acidic and better able to like nigrosine, Indian ink, or adhere to the sample. eosin blue to create a thick film over a slide surface. MICROBIOLOGY LAB LABORATORY 6: BACTERIAL STAINING Method: iii. Allow the smear to air 1. Prepare the lab bench by removing dry. Note: if the extraneous items and cleaning the inoculation on the slide surface with table disinfectant. is completely 2. Wipe a microscope slide. Clean with a transparent after drying, paper towel or Kim Wipe. add a few more 3. Using a grease pencil, draw a circle loopfuls of organism. about the size of a dime on the bottom 5. Regardless of the culture source, when of the slide. the smear is completely dry, heat-fix by 4. Transfer culture to the top of the quickly passing the smear over the slide, within the circle previously flame 2-3 times. drawn: 6. Repeat this smear prep procedure for the a. From the solid culture: remaining 3 cultures. i. Place 1 drop of water from the loop onto the center of the circle. ii. Transfer a small amount of solid inoculum into the drop of water and disperse it thoroughly. iii. Spread the liquid into a thin area about the size of a dime. iv. Allow the smear to air dry. b. From the liquid culture i. Place 3-4 loopfuls of liquid culture on the center of the circle. ii. Spread the suspension into a thin area about the size of a dime. MICROBIOLOGY LAB LABORATORY 6: BACTERIAL STAINING wash bottle. The smear will appear as SIMPLE STAIN PROCEDURES a purple circle on the slide. 1. Place the slide on the staining tray and 6. Decolorize using 95% ethyl alcohol or cover the smear with Methylene Blue acetone. Tilt the slide slightly and apply Dye for approximately 1 minute. ◦ the alcohol drop by drop for 5-10 2. While holding the slide at a 45 angle, seconds until the alcohol runs almost gently wash the surface with water 2-3 clear. Be careful not to over-decolorize. times. Allow the wash to drain into the 7. Immediately rinse with water. staining tray. 8. Gently flood with safranin to 3. Open the package of bibulous paper and counterstain and let stand for 45 place the wet slide inside. Close the seconds. package and carefully press on the cover 9. Tilt the slide slightly and gently rinse to blot the slide dry. Do not blot too with tap water or distilled water using a vigorously or the slide will crack. There wash bottle. is no need to remove paper from the 10. Blot dry the slide with bibulous paper. bibulous paper pad. 11. View the smear using a light-microscope 4. Once the slides are dry, examine them under oil immersion. microscopically. Be sure to use the 100x PRIMARY STAIN: Crystal Violet objective and oil. MORDANT: Gram’s iodine GRAM STAIN PROCEDURES DECOLORIZER: 95% ethyl alcohol or 1. Place the slide with a heat-fixed smear acetone then water on the staining tray. COUNTERSTAIN: Safranin 2. Gently flood the smear with crystal BLOT DRY violet and let stand for 1 minute. 3. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. 4. Gently flood the smear with Gram’s iodine and let stand for 1 minute. 5. Tilt the slide slightly and gently rinse with tap water or distilled water using a