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Negative Staining Technique Technical Training Centre Lund, Sweden FiS Q A TM-00031:11 2/9908 ppt 11 Negative Staining Technique Objective : To colour the background in order to provide a contrast against which to observe microbial cells which remain colourless (they not take up the stain) Reagent : 10% nigrosin in water Alternative : 0.45% methylene blue in 30 ml saturated alcohol mixed with 10% w/v potassium hydroxide in water Method : Place drop of negative stain solution on a clean glass slide Emulsify a small sample from an agar plate culture in the drop of stain solution and spread over a 1-2 cm2 area Allow to air dry Observe microscopically under oil immersion Note! Negative staining technique is a good alternative to investigate size and form of cells This technique is to prefer for the untrained microscopist or if no phase microscope is available With a good phase contrast microscope trained microbiologists prefer a wet preparation (some organismmaterial dispersed in water), a cover glass and then using the phase microscope with the 100 objective Most often size and shape can be viewed in the gram stain preparation But if this is difficult you can use negative staining or wet preparation Technical Training Centre 2/9908 FiS Q A TM-00031:11 11 Catalase Test Technical Training Centre Lund, Sweden FiS Q A TM-00031:12 2/9908 ppt 12 Catalase Test Reagent: 10 vol % H2 O2 Add drop of H2 O2 to the surface of one colony Positive result: Technical Training Centre 2/9908 Vigorous bubbling from surface of colony indicates the presence of the enzyme catalase which breaks down the hydrogen peroxide into water + oxygen gas, which is given off as bubbles FiS Q A TM-00031:12 12 Oxidase Test Kits are availablesafe and convenient Technical Training Centre Lund, Sweden FiS Q A TM-00031:13 2/9908 ppt 13 Oxidase Test Oxidase Reagents: 1% solution of tetramethyl-p-phenylenediamine dihydrochloride in distilled water Soak one filter paper with reagent and make a smear test of the bacteria culture or drop one drop of reagent solution onto colony on agar plate Positive result: If the bacteria are oxidase positive the colouring substance is oxidized into a purple blue colour The reagent is available in several forms * dry powder (mix with distilled water to form solution) * reagent kits available as sticks or plates/pads Note: Technical Training Centre 2/9908 Not recommended for use in wet or solution form since this reagent is carcinogenic FiS Q A TM-00031:13 13 Gram Differentiation Technique Technical Training Centre Lund, Sweden FiS Q A TM-00031:14 2/9908 ppt 14 Gram Differentiation Technique Objective : To validate the gram staining technique by means of a biochemical test which differentiates the physical differences, in cell wall structure between gram positive and gram negative cells Method : Place one droplet of 3% potassium hydroxide(KOH) on a microscope slide Take a needle of organism material from a colony on an agar plate Emulsify the organism material very well in the KOH solution After about 10 seconds, raise the needle from the emulsified material and look for the presence of long sticky threads between the needle and the slide Stop stirring if no threads are observed after 15-20 seconds Interpretation of result: •Gram positive bacteria not form threads •Gram negative bacteria will show sticky thread formation Note : This validation test is most useful in cases where the gram stain result is unclear, eg Coryneform group Technical Training Centre 2/9908 FiS Q A TM-00031:14 14 Gram Staining Technique (1) Technical Training Centre Lund, Sweden FiS Q A TM-00031:15 2/9908 ppt 15 Gram Staining Technique (1) Objective : To differentiate between cell types with different cell wall structures by means of a differential staining technique Method : Making a smear Place a drop of water on a clean glass slide Place a needle of microbial material from a colony on an agar plate in the water drop and mix/emulsify and spread over an area of approximately 1-2 square cm Allow the smear to air-dry Heat fix the smear to the slide by means of passing through the hot part of a flame 2-3 times Allow to cool Place the slide on a staining rack over a sink Technical Training Centre 2/9908 FiS Q A TM-00031:15 15 Gram Staining Technique(2) Technical Training Centre Lund, Sweden FiS Q A TM-00031:16 2/9908 ppt 16 Gram Staining Technique (2) Staining method Apply solution 1(Crystal violet) liberally to the smear Allow to stand for 30 seconds Rinse with water Apply solution (Gram’s iodine) Allow to stand for minute Rinse with water Apply decolourising solution 3(Acetone/alcohol) Allow to stand for only a few seconds before rinsing with water Apply solution (Safranin - red counter stain) Allow to stand for 30 seconds Rinse with water Gently pat dry with tissue/paper towel Examine microscopically under oil immersion Interpretation of result: Gram positive cells are coloured deep purple/blue Gram negative cells are coloured red Technical Training Centre 2/9908 FiS Q A TM-00031:16 16 Examples of colonial Morphology (1) Technical Training Centre Lund, Sweden Technical Training Centre 2/9908 FiS Q A FiS Q A TM-00031:17 2/9908 TM-00031:17 ppt 17 17 Examples of colonial Morphology (2) Technical Training Centre Lund, Sweden Technical Training Centre 2/9908 FiS Q A FiS Q A TM-00031:18 2/9908 TM-00031:18 ppt 18 18 Moulds and Yeasts Technical Training Centre Lund, Sweden Technical Training Centre 2/9908 FiS Q A FiS Q A TM-00031:19 2/9908 TM-00031:19 ppt 19 19 Fungi Technical Training Centre 2/9908 FiS Q A TM-00031:20 20 Laboratory Requirements Equipment Technical Training Centre Lund, Sweden FiS Q A TM-00031:21 2/9908 ppt 21 Laboratory Requirements Essential equipment: Autoclave (1 essential, convenient to have) for making / sterilising media for sterilising contaminated plates / media prior to disposal) Microscope (Binocular - must be of good quality e.g Zeiss, Leitz, Olympus, Nikon) Incubator (need at least 2) x 30°C operating temperature x 25°C x 55C pH meter Hot plate stirrer (for making media) Refrige- Trolleys (1 x small for laboratory A walk in refrigerator rator for storing ready made culture media etc is also useful/convenient (for transporting sample packs) (Water distillation equipment) Bunsen burner Top loading balanced scale (weighing media - only d-place necessary) Water bath and/or oven Technical Training Centre 2/9908 FiS Q A TM-00031:21 21