381 PHT Lab 2 1 2 Staining of

381 PHT Lab #2 1

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Staining of Bacteria cells are almost colorless and transparent A staining technique is often applied to the cells to color them → Their shape and size can be easily determined under the microscope. 3

Principle of staining Stains → combine chemically with the bacterial protoplasm. Commonly used stains are salts: v Basic dyes: colored cation + colorless anion e. g. methylene blue (methylene blue chloride) MB+ + Clv Acidic dyes: colored anion + colorless cation e. g. eosin ( Na+ + eosin-). 4

Bacterial cells are slightly negatively charged ( rich in nucleic acids bearing negative charges as phosphate groups) → combine with positively charged basic dyes Acidic dyes do not stain the bacterial cell → can stain the background material with a contrasting color. 5

Types of staining techniques Simple staining Differential staining (use of a single stain) (use of two contrasting stains separated by a decolorizing agent) For visualization of morphological shape & arrangement. Identification Gram stain Visualization of structure Acid fast stain Spore Capsule stain 6

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Smear Preparation: Preparation and Fixation of Bacteria for Staining. Objective: To kill the microorganism & fix them to the slide to prevent them from being washed out during the process of staining. 8

Smear preparation S Fixation 9

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Definition: It is the use of single basic dye to color the bacterial organism. e. g. methylene blue, crystal violet, safranin. All bacteria take the color of the dye. Objective: To show the morphological shapes and arrangement of bacterial cells. 11

Simple Staining Procedure: - MB 1 -2 min 12

Basic Shapes of Bacteria Cocci Bacilli 13

Arrangements Cocci Irregular Clusters Staphylococci Tetrads Micrococci Chains or Pairs Streptococci 14

Results Type of staining: Name of stain: Shape of cells: Arrangement of cells: Color: Name of m. o: 15

Simple Staining Type of staining: - Simple Stain Name of dye: - Methylene blue Shape of cells: - bacilli Arrangement of cells: strain Color: - Blue Name of m. o: - Bacillus 16

Simple Staining Type of staining: - Simple Stain Name of dye: - Methylene blue Shape of cells: - cocci Arrangement of cells: clusters Color: - Blue Name of m. o: Staphylococci 17

Simple Staining Type of staining: - Simple Stain Name of dye: - Crystal violet. Shape of cells: - cocci Arrangement of cells: clusters Color: - Purple Name of m. o: Staphylococci 18

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Gram Stain: It is the most important differential stain used in bacteriology because it classified bacteria into two major groups: a)Gram positive: Appears violet after Gram’s stain b) Gram negative: Appears red after Gram’s stain 20

Crystal violet ↓ Iodine ↓ Alcohol ↓ Safranin 21

Gram +ve S. aureus Gram –ve E. coli Step 1: Crystal Violet Step 2: Gram’s Iodine Step 3: Decolorization (Aceton-Alcohol) Step 4: Safranin Red 22

Step 1: Crystal Violet Step 2: Gram’s Iodine Step 3: Decolorization (Aceton-Alcohol) Step 4: Safranin Red 23

Gram’s +ve Bacteria Gram’s -ve Bacteria 24

Gram’s +ve Bacteria Gram’s -ve Bacteria 25

Gram-positive bacteria Have a thick peptidoglycan layer surrounds the cell. The stain gets trapped into this layer and the bacteria turned purple. Retain the color of the primary stain (crystal violet) after decolorization with alcohol Gram-negative bacteria have a thin peptidoglycan layer that does not retain crystal violet stain. Instead, it has a thick lipid layer which dissolved easily upon decoulorization with Aceton-Alcohol. Therefore, cells will be counterstained with safranin and turned red. 26

Gram Stain Materials: Cultures of Staphylococcus aureus, Candida albican, Bacillus subtilis, E. coli Gram stain: Crystal violet (primary stain) Gram’s iodine (mordant) Acetone-alcohol (decolorizing agent) 27 Safranin (counter stain)

Gram Stain Procedure: safranin CV iodine s 30 sec 30 -60 sec 10 sec 2 min 28

Results: Shape: Cocci Arrangment: irregular clusters Colour: Violet Gram’s reaction: Gram’s +ve Name of microorganism: Staphylococci 29

Results: Shape: Oval Arrangment: Single Colour: Violet Gram’s reaction: Gram’s +ve Name of microorganism: Candida 30

Results: Shape: Bacilli Arrangment: Chains Colour: Violet Gram’s reaction: Gram’s +ve Name of microorganism: Bacillus 31

Results: Shape: Rods Arrangment: Single Colour: red Gram’s reaction: Gram’s –ve Name of microorganism: Gram negative bacilli 32

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Negative staining (Indirect staining with acidic dye) The negative staining technique does not stain the bacteria due ionic repulsion. but stain the background. The bacteria will appear colorless against a dark background. No heat fixation or strong chemicals are used→ the bacteria are less distorted than in other staining procedure. Example: Nigrosine 34

Negative staining Candida 35

Negative staining Staphylococci 36

Negative staining Bacillus 37

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