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1 381 PHT Lab #2. 2 3 Staining of Bacteria Bacteria cells are almost colorless and transparent A staining technique is often applied to the cells to.

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Presentation on theme: "1 381 PHT Lab #2. 2 3 Staining of Bacteria Bacteria cells are almost colorless and transparent A staining technique is often applied to the cells to."— Presentation transcript:

1 1 381 PHT Lab #2

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3 3 Staining of Bacteria 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. Their shape and size can be easily determined under the microscope.

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

5 5 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.

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

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9 9 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. To kill the microorganism & fix them to the slide to prevent them from being washed out during the process of staining.

10 10 Smear preparation S Fixation

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13 13 Definition: It is the use of single basic dye to color the bacterial organism. It is the use of single basic dye to color the bacterial organism. e.g. methylene blue, e.g. methylene blue, crystal violet, crystal violet, safranin. safranin. All bacteria take the color of the dye. All bacteria take the color of the dye.Objective:- To show the morphological shapes and arrangement of bacterial cells. To show the morphological shapes and arrangement of bacterial cells.

14 14 Simple Staining Procedure:- MB 1-2 min

15 15 Basic Shapes of Bacteria Cocci Bacilli

16 16 Arrangements Cocci Irregular ClustersChains or PairsTetrads Staphylococci MicrococciStreptococci

17 17Results Type of staining: Name of stain: Name of stain: Shape of cells: Shape of cells: Arrangement of cells: Color: Name of m.o:

18 18 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

19 19 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 Staphylococci

20 20 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 Staphylococci

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23 23 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

24 24 Crystal violet ↓Iodine↓Alcohol↓Safranin

25 25 Gram +ve S.aureus Gram –ve E.coli E.coli Step 1: Step 1: Crystal Violet Step 2: Gram’s Iodine Step 3: Decolorization (Aceton-Alcohol) (Aceton-Alcohol) Step 4: Safranin Red

26 26 Step 1: Step 1: Crystal Violet Step 2: Gram’s Iodine Step 3: Decolorization (Aceton-Alcohol) (Aceton-Alcohol) Step 4: Safranin Red

27 27 Gram’s +ve Bacteria Gram’s -ve Bacteria

28 28 Gram’s +ve Bacteria Gram’s -ve Bacteria

29 29 Gram-positive bacteria Have a thick peptidoglycan layer surrounds the cell. 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.

30 30 Gram Stain Materials:- Cultures of Staphylococcus aureus, Candida albican, Candida albican, Bacillus subtilis, Bacillus subtilis, E.coli E.coli Gram stain: Crystal violet (primary stain) Crystal violet (primary stain) Gram’s iodine (mordant) Gram’s iodine (mordant) Acetone-alcohol (decolorizing agent) Acetone-alcohol (decolorizing agent) Safranin (counter stain) Safranin (counter stain)

31 31 Gram Stain Procedure: s CV iodine sec 2 min 10 sec safranin 30 sec

32 32 Results: Shape: Cocci Arrangment: irregular clusters Colour: Violet Gram’s reaction: Gram’s +ve Name of microorganism: Staphylococci

33 33 Results: Shape: Oval Arrangment: Single Colour: Violet Gram’s reaction: Gram’s +ve Name of microorganism: Candida

34 34 Results: Shape: Bacilli Arrangment: Chains Colour: Violet Gram’s reaction: Gram’s +ve Name of microorganism: Bacillus

35 35 Results: Shape: Rods Arrangment: Single Colour: red Gram’s reaction: Gram’s –ve Name of microorganism: Gram negative bacilli Gram negative bacilli

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38 38 Negative staining (Indirect staining with acidic dye) The negative staining technique does not stain the bacteria due ionic repulsion. but stain the background. 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

39 39 Negative staining Candida

40 40 Negative staining Staphylococci

41 41 Negative staining Bacillus

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