Basic Microbiology and Immunology Practical Course

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1 Basic Microbiology and Immunology Practical Course

2 2 Lab # 2: Colouring the microorganisms

3 Rules that must be followed to maintain an aseptic zone 3 For most bacterial cultures, you will use a sterile loop to inoculate or to obtain an inoculum. Flame the loop to red-hot prior to use to sterilize it. Cool the loop prior to touching the culture.

4 4 Results of Sources of Contamination

5 5 Fungi Bacteria

6 Bacterial colony morphology 6 A bacterial colony: Macroscopically visible cluster of bacteria growing on the surface of or within a solid medium, presumably cultured from a single cell. Plate with nine (9) different colonies identified

4- Margin: (Entire- irregular). 5- Surface: (Smooth-rough).

7 Results 7 1- Shape: (Circular-irregular). 2- Size: -Small (pinpoint). -Medium (2-5 mm). -Large (>5 mm). 3- Color: state the color you see. 4- Margin: (Entire- irregular). 5- Surface: (Smooth-rough). 6- Optical characters: (opaque- translucent). 7- Elevation: (Flat- elevated). 8- Texture: (Soft- hard- mucoid). Elevated

8 Microscopy 8

9 Staining 9 Even with the microscope, bacteria are difficult to see unless they are treated in a way that increases contrast between the organisms and their background. The most common method to increase contrast is to stain part or all of the microbe.

10 Preparing heat fixed bacterial A heat fixed smear: smear is a thin layer of the bacterial specimen dried and fixed onto the slide. 10 Procedure: 1- A circle should be marked on the under side of a slide with a permanent marker. Glass slide

culture (after shaking), place directly on the slide and spread gently in 1 cm

11 Preparing heat fixed bacterial 2- To prepare a smear from a suspended culture, by means of an inoculating loop, aseptically transfer 3-4 loopfuls of the culture (after shaking), place directly on the slide and spread gently in 1 cm area. 3- Air dry or dry over the flame. smear 11 Inoculation loop Dried bacterial smear

Preparing heat fixed bacterial 4- Heat fixation : By

12 Preparing heat fixed bacterial 4- Heat fixation : By passing the slide about five passes through the flame. Heat fixation accomplishes three things: (1) kills the organisms. (2) Causes the organisms to adhere to the slide. (3) Alters the organisms so that they more readily accept stains (dyes). smear 12 5 to 6 times

13 Precautions during heat fixation 13 If the slide is not completely dry when you pass it through the flame, the organism will be boiled and destroyed. If you heat-fix too much, the organisms may be incinerated, and you will see distorted cells and cellular remains. Fixation while Incomplete drying Or Too much fixation If you heat-fix too little, the organism may not stick and will wash off the slide in subsequent steps. No Film Too little fixation

14 Types of dyes (stain) Stains: are chemicals containing chromophores (groups that impart color). Their specificity is determined by their chemical structure and charge they carry. Accordingly there are 3 types of dyes Basic dye (cationic dye): positively charged chromophore e.g. crystal violet, safranin, basic fuchsin & methylene blue. Acidic dye (anionic dye):negatively charged chromophore e.g. nigrosine & sodium eosinate. Neutral dye: Has both charged chromophore (net charge is neutral). e.g. eosin methylene blue. 14

15 Staining techniques 15 Non-differential stain Differential stain Structural stain Direct Gram stain Spore stain Negative Acid fast stain Capsule stain

16 Non-differnetial simple stain Uses One dye 16 Direct stain Direct Negative stain Negative Cationic dye +ve (Crystal violet) Bacterial cell Anionic dye -ve (Negrosine) Attraction -vely charged surface Repulsion Colored MO. # colorless background Colorless MO. # colored background

17 Direct simple stain procedure Prepare a heat fixed bacterial smear as previous. 2. Leave to cool. 3. Using a dropper, cover the film with crystal violet or safranin. 4. Leave for 30 sec in case of using crystal violet, 3-5 min for safranin.

18 18 NB: Oil must be cleaned from the oil immersion lens using organic solvent: xylene, after finishing sample examination

Escherichia coli Name of stain Direct stain Direct stain Type of stain

19 Microscopic examination 19 Name of M.O Cocci e.g. Staphylococcus aureus rods e.g. Escherichia coli Name of stain Direct stain Direct stain Type of stain Non-differential simple Non-differential simple Shape Cocci Bacilli or rods Arrangement Clusters Single Size Small Small Color Violet Red

20 Indirect Stain: Negative stain 20 Procedure: 1.Prepare an air dried bacterial smear 2.Add one drop of nigrosin on the side of the slide 3. Holding a second slide at a 45 degree angle, allow the drop to spread along the angled slide. 4. Allow the dye to thoroughly air dry. 5. DO NOT WASH!!! 6. Apply immersion oil to the smear and observe under light microscope.

21 Negative stain technique 21

22 Negative Stain: Staphylococcus aureus 22 Name of Microorganism Name of Stain Type of stain Shape Size Arrangment Color Cocci e.g. Staphylococcus aureus Indirect negative Stain Non- differential simple Cocci Small Cluster Colorless

23 Negative Stain: Rods 23 Name of Microorganism Name of Stain Type of stain Shape Size Arrangment Color Gram Negative Rods Indirect negative Stain Non- differential simple Bacilli or rods Small Single Colorless

24 24 See you next lab With new techniques & more bacterium

Steps of microbial smear preparation :

Steps of microbial smear preparation : Lab 4 STAINING Practical Microbiology Microbial smear : It is a very small amount of microbial growth ( broth or solid ) spreaded on a clean slide and drying by air. Fixation : The process of passing the