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Microbiology Lab (3)

Microbiology Lab (3). Differential Stains (Gram stain & Acid Fast Stain) Abdelraheem BA. Objectives. To learn how to stain bacteria with gram stain & acid-fast stain. To determine the form, gram reaction, shape and arrangement of stained bacteria. Differential stains.

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Microbiology Lab (3)

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  1. Microbiology Lab (3) Differential Stains (Gram stain & Acid Fast Stain) Abdelraheem BA

  2. Objectives • To learn how to stain bacteria with gram stain & acid-fast stain. • To determine the form, gram reaction, shape and arrangement of stained bacteria.

  3. Differential stains • A stain that is used to differentiate between certain groups of bacteria. • This techniques requires the use of: • At least 3 chemical reagents. • Heat fixation of prepared bacterial smears. • Examples: • Gram stain: Gram +veVs Gram –vebacteria. • Acid-fast stain: Acid fast Vs Non-Acid fast.

  4. Gram stain • A technique that is used to stain bacteria and yeast. • One of the most commonly performed procedures in microbiology lab. • A very important step in the identification of bacteria.

  5. Reagents in Gram stain • Primary stain: Crystal violet. • Mordant*: Gram’s Iodine. • Decolorizing agent: 95% Alcohol or acetone-alcohol (50:50). • Counter stain: Safranin • *Mordant: • A substance that forms a colored insoluble complex by binding the primary. • CV-I complex hardly leave the cell. • Result: intensified color & fixation of stain.

  6. Steps to perform gram stain • Allow the smear to completely air dry. • Heat fixation. • Place the slide on the staining tray. • Flood the smear with the primary stain (Crystal violet) for 1 minute. • Wash the smear with tap water to remove extra stain. • Flood the smear with iodine for 1 minute. • Wash the smear with tap water to remove extra iodine.

  7. Steps – Cont’d • Rinse with the decolorizing agent (a crucial and a sensitive step); • Few drops for 10 seconds. • Immediately wash the slide with tap water, to remove extra stain. • Flood the smear with the counter stain (Safranin) for 1 minute. • Wash with tap water. • Use bibulous paper to dry the stained smear and examine with light microscope.

  8. What is the principle? • Bacterial cell wall review: • G+ve: • Thick layer of peptidoglycan. • Thin layer of lipids. • Lipids will be dissolved by alcohol (forming minute pores). • These pores will then shrink by the dehydrating effect of alcohol. • CV-I complex will be prevented from leaving. • G-ve: • Thin layer of peptidoglycan. • Thick layer of lipids. • Due to alcohol, lipids will dissolve forming large pores. • CV-I complex will leave, the bacteria become colorless.

  9. Bacterial cell wall

  10. Gram Stain - Summary

  11. Precautions • Decolorization is a crucial step; • Over decolorization; loss of primary stain (False gram –ve bacteria) • Insufficient decolorization; failure to remove CV-I complex (Fals gram +ve bacteria). • Fresh cultures must be used; • Old cultures (in case of gram +ve bacteria); damage to cell wall which leads to failure to retain the CV-I complex. • Gram variable result. • Suitable fixation must be used; • Too much heat will destroy cell wall; • Gram +ve bacteria will lose the primary stain (False gram –ve bacteria). • Freshness of reagents.

  12. Acid Fast stain • Most bacteria are stained by gram stain. • Members of genus Mycobacterium and Nocardia have a special cell wall. • Thick waxy layer (Lipoidal). • This makes these bacteria resistant to many antiseptics and antibiotics. • This is the reason for being ACID FAST.

  13. Mycobacteria • Cell wall is composed of: • Fatty acids (mycolic acid). • Complex waxes. • Complex unique glycolipids. • Examples: • M. tuberculosis; the causative agent of TB. • M. leprae; the causative agent of leprosy.

  14. Membrane of Mycobacteria

  15. What does “Acid Fast” mean? • The cell wall keeps the color red of Carbolfuchsin (the primary stain), when decolorized with acid-alcohol. • Other bacteria will be decolorized and stain blue, the color of Methylene blue (the counter stain).

  16. Reagents of Ziehl-Neelsen Method • Primary Stain. • Carbolfuchsin; a phenolic dye and soluble in the waxy cell wall. • Used with the aid of heating (figure). • After this step, all bacteria will stain red. • Decolorizer. • Acid-alcohol. • Before addition, the slide must be cooled to harden the waxy cell wall. • After this step. • Acid fast bacteria will remain red. • Non-Acid fast bacteria will be colorless. • Counter stain. • Methylene blue. • Acid fast bacteria remain red. • Non-Acid fast bacteria will stain blue.

  17. Good Luck

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