Techniques of identification of bacteria

This Slide contains the information about Techniques used for the identification of bacteria and also contains Various methods used to stain the bacteria.
Positive Staining
negative staining
Grams Staining
Acid Fast Staining

1. Techniques Of Identification Of Bacteria By Mr.Rushikesh Laxman Tamhane

2. Introduction Staining is an auxiliary technique used in microscopy. Stains and dyes are frequently used in biology and medicine to highlight structure in biological tissues. Stains may be used to define and examine bulk tissues, cell populations or organelles with individual cells. Bacteria are microscopic organisms. They are mostly colorless and to visualize them to study their structure, shape and other structural characteristics.

3. Types Of Stains ACIDIC :- Negatively charged acid radicals imparts color in eosin, acid fuchsine, malachite green, Indian ink. BASIC:- Positively charged basic radicals combines with negatively charged particles in cytoplasm and gives color. e.g., Haematoxillin , Methylene blue, Crystal violet. NEURTAL:- Both positively and negatively charged imparts different colors to different components. e.g., Geimsa’s stain and Leishman’s stain.

4. STAINING TECHNIQUES POSITIVE STAINING Where the actual cells are themselves colored and appear in clear background. Simple staining: A stain which provides color contrast but gives same color to all bacteria an cells. e.g., Malachite green and Crystal violet. Differential staining: A stain which imparts different colors to different bacteria is called differential stain (which contains more than one stain) e.g., Gram’s stain, Acid fast staining and Endospore staining.

5. Staining Techniques NEGATIVE STAINING : Where the cells remain clear (uncolored) & the background is colored to create a contrast to aid in the better visualization of the image. Nigrosin Indian Ink

6. Requirements:- Loefflers Methylene Blue Dilute Carbon Fuschin Distilled Water Compound microscope Cedar wood Oil Fixed Smear Simple Staining

7. Procedure Make a thin smear on a slide. Heat fixes the smear by passing the slide 2-3 times gently over the Bunsen flame with the smear side up. Pour Loeffler’s Methylene blue over the smear and allow it to stand for 3 minutes. Wash the stained smear with water and air dry it. Observe the smear first under low power (10X) objective, and then under oil immersion (100X) objective. Observe the presence of organisms and also the cellular content of sample.

8. Result Of Simple Staining Spherical Rod Shaped

9. HANS CHRISTIAN JOACHIM GRAM T he Gram stain was devised by the Danish physician, Hans Christian Joachim Gram, while working in Berlin in 1883.He later published this procedure in 1884.At the time, Dr. Gram was studying lung tissue sections from patients who had died of pneumonia.

10. Gram Staining Gram staining is most widely used differential staining in microbiology. It differentiates the bacteria into two groups: Gram positive and Gram negative. Gram Positive Bacteria :Gram-positive bacteria retains the gram stain and show a visible violet colour upon the application of mordant (iodine) and ethanol (alcohol). Gram Negative Bacteria: The gram-negative bacteria are stained by a counterstain such as safranin, and they are de-stained because of the alcohol wash. Hence under a microscope, they are noticeably pink in colour.

11. Components Used Primary Stain:- The first reagent is called the primary stain. Its function is to impart its color to all cells. Mordant:- The second stain is a mordant used to intensify the color of the primary stain. Decolorising Agent:- To establish color contrast Decolorising agent is used

12. Procedure It consists of four steps: Primary staining: Th e smear is covered Crystal Violet, for 1 minute and washed with water. Mordanting : It is then covered with Gram’s Iodine, kept for 1 minute, and washed with water. Decolorization : T he smear is covered with alcohol and is washed with water immediately. Counter staining : Th e smear is then covered with safarnin, kept for 30 seconds and washed with water. Observe under microscope.

13. Result Bacteria that manage to keep the original purple dye have only got a cell wall-they are called Gram-positive. Bacteria that lose the original purple dye and can therefore take up the second red dye have got both cell wall and cell membrane-they are called Gram-negative .

14. Acid Fast Staining The acid fast staining is a modification of Ehrlich’s (1882) method also known as Ziehl-Neelsen stain. It is used for bacilli belonging to the genus Mycobacterium especially Mycobacterium tuberculosis, Mycobacterium laprae and also for Nocardia. Basic requirements: Primary and mordant staining with strong carbol fuchsin (red). Decolorization with acid alcohol. Counterstain with methylene blue.

15. Acid Fast & Non Acid Fast Bacteria Acid Fast Bacteria:- They doesnt decolorize by acid after staining and gives Pink or Red color Non Acid Fast Bacteria:- They easily decolorize by acid after staining and gives Blue Color.

16. Procedure Make a smear. Air dry. Hear fix. Flood smear with Carbol-Fuchsin stain. Steam for 5 minutes. Add more of the stain as needed. Cool slide for 5 minutes. Wash with distilled water. Flood with acid alcohol (leave for 15 seconds). Tilt the slide 45⁰ over the sink and add acid alcohol drop wise until the red color stops streaming from the smear. Rinse with distilled water. Add Loeffler’s methylene blue stain (Counter stain). Leave the stain on smear for 15-20 seconds. Rinse the slide and let it dry. Use oil immersion objective to view.

17. Result The stained smear contains pink colored slender rod shaped structures with curved ends. Smear is Positive for Acid Fast Bacilli.