STAINING TECHNIQUES oS 7 ( 2 aINTRODUCTION: » Bacteria are microscopic organisms. They are also colorless for the most part. In order to visualize them to study their structure, shape and other structural characteristics, it becomes necessary to make them more easily visible. This means that the structures have to be contrasted from their environment so that they can be seen easily. STAIN: Stain is a dye used to color the living or dead organelles.— __ 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. S— * NEURTAL Both positively and negatively charged imparts de colors to different components. © e.g., Geimsa's stainand Leishman’sstaid. @~~ STAINING TECHNIQUES POSITIVE STAINING — Where the actual cells are themselves colored and appear in a 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. A ~+ 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.BACTERIAL SMEAR PREPARATION: Smear - isa distribution of bacterial cells on a slide for the purpose of viewing them under the microscope. Method: -Aseptically a small sample of the culture is spread over a slide surface. “This is then allowed to air dry. -The next step is heat fixation to help the cells adhere to the slide surface. -The smear is now ready for staining.SIMPLE STAINING LOEFFLER’S METHYLENE BLUE: It is generally the most useful, it shows the characteristic morphology of polymorphs, lymphocytes and other cells more clearly than do stronger stains such as the Gram stain or dilute carbol fuchsin. % POLYCHROME METHYLENE BLUE: This is made by allowing Loeffler’s methylene blue to ‘ripen’ slowly. The slow oxidation of the methylene blue forms a violet compound that gives the stain its polychrome properties.The ripening takes 12 months or more to complete, or it may be ripened quickly by the addition of 1.0% potassium carbonate (K2co3) to the stain. It is also employed in McFadyean’s reaction. Incontrast to the blue staining of most structures by the methylene blue, the violet component stains acidic cell structures red-purple, e.g. the acid capsular material of the anthrax bacillus in the McFadyean reaction. DILUTE CARBOL FUCHSIN Made by diluting ZiehI-Neelsen’s stain with 10-20 times its volume of water. Stain for 10-25 seconds and wash well with water. Over-staining must be avoided, as this is an intense stain, and prolonged application colours the cell protoplasm in addition to nuclei and bacteria.— wy * ~ REQUIREMENTS + Loefflers methylene blue + Dilute Carbol Fuchsin + Distilled water + Compound microscope + Cedar wood oil + Fixed smear— — a 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 oll immersion (100X) objective. Observe the presence of organisms and also the cellular content of sample. uy g) . _s \ ae J ” ee | SS wes! 5 v9 a8 Pd Hays,— ww Y 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: They have a thick cell wall of peptidoglycan and other polymers Peptidoglycan consists of interweaving filaments made up of alternating N-acetylmuramic acid and N-acetylglucosamine monomers. a) re g— — ~ of phospholipid and bacterial lipopolysaccharides outside of their thin peptidoglycan layer. * The outer membrane protects gram negative bacteria againsts penicillin and lysozymes. * Gram Negative Bacteria: They have an outer membrane ~—< aa ___ PROCEDURE OF GRAM STAINING + It consists of four steps: »Primary staining: the smear is covered Crystal Violet, for 1 minute and washed with water. >Mordanting: it is then covered with Gram’s lodine, kept for 1 minute, and washed with water. > Decolorization: the smear is covered with alcohol and is washed with water immediately. >Counter staining: the smear is then covered with safarnin, kept for 30 seconds and washed with water. vu » Observe under microscope. CyQ RESULT * Bacteria that manage to keep the original purple dye have wh wt Ion 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.— aor _/~ ACID-FAST STAINING + The acid fast staining is a modification of Ehrlich’s (1882) method also known as Ziehl-Neelsen stain. + Itis used for bacilli belonging to the genus Mycobacterium especially Mycobacterium tuberculosis, Mycobacterium laprae and also for Nocardia. + Acid fastness of the acid-fast bacilli is attributed to the presence of unsaponifiable wax fraction called mycolic acid in their cell wall. + Basic requirements: ¥ Primary and mordant staining with strong carbol fuchsin (red). ¥ Decolorization with acid alcohol. ¥ Counterstain with methylene blue. \_/ v— __/* © PROCEDURE en § C 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 45° over the sink and add acid alcohol drop wise until the red color stops streaming from the smear. Rinse with distilled water. ‘Add Loeffer’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. 9@ RESULT * The stained smear are contains pink colored slender rod shaped structures are seen with curved ends. + The smear is positive for acid fast bacilli.—, J” CAPSULE STAINING “ + The capsules serves as protective material by slowing down or preventing penetration of chemicals and body juices. PRINCIPLE * Chemically, the capsular material is a polysaccharide, a glycoprotein or a polypeptide. + Capsule staining is more difficult then other types of differential staining procedures because he capsular material are water soluble and may be dislodged and removed with vigorous washing. + The capsule is non-ionic, so that the dyes commonly used will not bind to it. Two-dyes,/one acidic and one/.— PROCEDURE — —_ + For positive staining of smears: + Make a smear from colony of S.pneumoniae on a clean grease free glass slide, and allow it to air dry. + Flood the smear with Crystal Violet and allow it to stain for 5-7 minutes. + Wash the smear with 20% copper sulphate solution and dry it. + Observe the smear first under low power(10X) objective, and then under oil immersion (100X) e@ objective. — + In the culture smear, the capsule is seen as a light blue in contrast to the dean sefole éolor of the cell. J— For negative staining of smears: ~ Take a clean grease free glass slide. * Put a large loopful of undiluted Indian ink on the slide. * Then add a small loopful of liquid bacterial culture to the Indian ink and emulsify. + Take a clean, grease free cover slip and place on the ink drop and press it down, so that the flim becomes very thin and thus pale in color. * Observe the wet flim under high power (40X) objective. + The capsule in negative staining method is seen as clear refractile around thé ofwehism(against a black. ou ~—A — Y ENDOSPORE STAINING + Spores are highly resistant inactive forms. * The morphology of bacterial endospores is best observed in unstained wet flims under the phase contrast microscope, where they appear as large , refractile, oval or spherical bodies within the bacterial mother cells or else free from the bacteria. + Different staining are available for staining of spores. Ley— — __/» ~~ © PROCEDURE + Flims are dried and fixed with minimal flaming. + Place the slide over a beaker of boiling water, resting it on the rim with the bacterial flim uppermost. + When, within several seconds, large droplets have condensed on the under side of slide, flood it with 5% aqueous solution of Malachite green and leave it for 1 minute while the water continues to boil. + Wash in cold water. + Treat with 0.5% safranin and 0.05% basic fuchsin for 30 seconds. aa + Wash and dry. 4 * This method colors the spores te and oxegettee) pean red. y) 0