Bacteriology revision Nada Mohamed Ahmed, MT (ASCP)i Types of Microscopes FunctionUsed for 1.Compound Light Microscope study the cells that are between 1 and 100 m. (what we use most often) Bacterial morphology 2-Stereoscopes also known as dissecting scopes Gives a three dimensional view (3D) of an object insects and leaves 3. Electron Microscopes revealed many organisms and sub cellular structure that were impossible to resolve with the light microscope Mitochondria,golgi apparatus Parts of microscope: Parts of microscope Function Eyepiece (ocular) The lens the viewer looks through to see the specimen. The eyepiece usually contains a 10X or 15X power lens. B-Body tube (Head) The body tube connects the eyepiece to the objective lenses Stage clipsMetal clips that hold the slide in place. Nosepiece: A rotating piece that holds the objective lenses Objective lenses: One of the most important parts of a compound microscope, as they are the lenses closest to the specimen. Arm(neck) The arm connects the body tube to the base of the microscope. Used to carry StageThe flat platform where the slide is placed. diaphragm: Adjusts the amount of light that reaches the specimen Stage ControlThese knobs move the stage left and right or up and down. Coarseadjustment- Fine adjustment: Brings the specimen into general focus. K-Fine adjustment: Fine tunes the focus and increases the detail of the specimen. Light source: The hole in the middle of the stage that allows light from the illuminator to reach the specimen. Base The base supports the microscope used to carry microscope Types of culture media Based on their consistency mediaExamplePicture Solid media contains 2% agar Nutrient agar, Blood agar Semi solid medium 0.5% agar. Motility medium Liquid media no agar. Nutrient broth Special media Substances contained Example Enriched media Substances like blood, serum, egg are added to the basal medium Blood agar, Chocolate agar Enrichment media Media. Liquid media is incorporated with inhibitory substances to suppress the unwanted organism Selenite F Broth Alkaline Peptone Water Selective media solid media The inhibitory substance is added to a solid media. Mac Conkeys medium for gram negative bacteria TCBS for V.cholerae L J medium M.tuberculosis Wilson and Blair medium S.typhi Potassium tellurite medium Diphtheria bacilli Indicator media These media contain an indicator which changes its colour when a bacterium grows in them. Blood agar Mac Conkeys medium Christensens urease medium Differential media media which has substances incorporated in it enabling it to distinguish between bacteria. Mac Conkeys medium Distinguish between lactose fermenters & non lactose fermenters. Sugar media Media containing any fermentable substance. Contain a small tube (Durhams tube) for the detection of gas by the bacteria. Usual Sugar media Transport media Media used for transporting the samples non nutrient soft agar gel containing a reducing agent Stuarts medium Special media Picture Enriched media Blood agar Chocolate agar Enrichmen t media Selenite F Broth Selective media Mac Conkeys medium Indicator media Christensens urease medium Differentia l media Mac Conkeys medium Sugar media Usual Sugar media Transport media Stuarts medium Classification of stain (Based on function of stain: StainFunctionExample Simple staining To study morphology and arrangement of bacteria. only one dye is used- differentiation among bacteria is impossible Crystal violet, Methylene blue, Basic fuschin, Malachite green Differential staining more than one dye is used- Differentiation among bacteria is possible it provides more information about the characteristics of the cell wall (Thickness). Grams staining, Acid-fast staining. Special staining more than one dye used -Special structures are seen. More than one dye used - Special structures are seen. Capsule staining, Spore staining. Basic requirements for staining RequirementsFunctionPicture Clean grease-free slide.To hold the smear Bacteria to be stained Inoculating loops(wire loop - Platinum loop)- to transfer bacterial suspension to slide Bunsen burner To sterilize inoculating loops before and after smear preparation. Pencil marker to markto mark ReagentsFunctionTime CRYSTAL VIOLET Primary stain Violet colored, stains all microorganisms sec 2.GRAM IODINE Mordant Forms Crystal violet iodine complexes bacteria sec 3.DECOLORIZER Acetone + Methanol Removes Crystal violet iodine complex from thin peptidoglycan layers sec 4.Gram safranin; Counter stain Red colored Stains thin walled Gram negative organism. 2 min REAGENTS USED IN GRAM STAIN ReagentsFunctionTime Carbol Fuchsin stain a lipid soluble, phenolic compound, which is able to penetrate the cell wall 10 minutes acid alcoholdecolorizerleave 15 seconds Loefflers Methylene Blue stain (Counter stain). This stain adds blue color to non-acid fast cells. 1 minute Reagents used in Ziehl- Neelsen Stainingprinciple Basic stain(+ve charge) To stain -ve charged molecules of bacteria Mostly used because cell surface is ve charge As cell surface is ve charged- Basic dyes mostly used. SIMPLE STAININGAll bacteria in smear take stain and appear in colour of stain. Capsule stain Negative Staining Capsule is not stain by Indian ink or Nigrossin instead the background is stain dark (black) Background leaving the capsule as unstained area (with hollow) colorless area. Gram staining Gram staining is used to determine gram status to classify bacteria broadly. It is based on the composition of their cell wall Ziehl-Neelsen stain Mycobacteria, which do not stain well by Gram stain, are stained with carbol fuchsin combined with phenol. In the hot Zn technique, the phenol-carbol fuchsin stain is heated to enable the dye to penetrate the waxy mycobacterial cell wall. Staining principle