Bacteriology-lab-Smears PDF

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Bacteriology LabBacterial smear preparationMicroscopic examinations may require examining specimen in a fixed state. Examination through smearsDifferent types of smear preparations are made as to the different type of sampleDone by fixing sediments, sample parts or imprints on a glass slide for dete...

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Bacteriology Lab Bacterial smear preparation Microscopic examinations may require examining specimen in a fixed state. Examination through smears Different types of smear preparations are made as to the different type of sample Done by fixing sediments, sample parts or imprints on a glass slide for detection and evaluation of microbial presence

Types of smear preparation Emulsification- commonly prepared on bacteria from a culture medium. From a solid media 1 drop or 2 loopsful of NSS is placed on the center of a sterile glass slide A small amount of colony is then scraped using a sterile loop and mixed on the NSS on the slide. Streaking- made using a sterile swab or an applicator stick, material is applied to the sterile glass slide rapidly in a direct or zigzag line throughout the slide. Best for gram staining of vaginal discharges and purulent wounds Spreading- performed by selecting a small part of the material and then placing it to a sterile glass slide and gently teasing the mucus strands apart by using an applicator stick or inoculating loop. This method is best for mucopurulent materials like sputum

Pull apart- achieved by placing a drop of specimen on a sterile slide, covered by another sterile slide, and the two slides are pulled apart in an uninterrupted motion. Best for blood smears, bone marrow aspirates and enzymatic lavage. Wedge type/Film- prepared by placing a drop of blood on one end of a slide and then dispersed using a spreader slide (coverslip) over the glass slides length Commonly used on blood specimens for detection of blood parasites like malaria Touch preparations/Impression smearaccomplished by touching the tissue or specimen using a glass slide, allowing the cells to transfer directly to the slide for examination. Best for collecting urethral discharges, excised tissues and smears from skin slits Looping out- only made for sputum specimens used for Acid Fast Smears A small part of the sputum is smeared on 1/3 of a slide by a looping manner until dried. Smear should have a size of 2x3cm

Simple staining Defined as the process of artificially imparting color or dye to the organisms. Considered the simplest method of staining Utilizes only one type of stain and its color shall be imparted to the organism

May not allow us to visualize special structures of bacterial cells  Endospores  Flagella  Metachromatic granules

- have a thinner peptidoglycan layer

Several dyes can be used for simple staining  Methylene blue  Safranin  Crystal Violet

All cocci are Gram positive except for Neisseria Veiloneilla Branhamella All bacilli are Gram negative except for Bacillus Clostridium Corynebacterium Erysipelothrix Lactobacillus Listeria Nocardia Kurthia Rothia Actinobacillus

Methylene blue is an effective stain since most bacteria are stained when an alkaline dye permeates the cell wall and adheres by weak ionic bonds to the negative charges of the bacterial cell

Gram Staining Is a type of differential staining A staining technique in which the primary purpose is to be able to classify the microorganism into two major groups Bacteria can be differentiated into two major groups, since crystal violet retention will be depend on the cell wall composition Difference in composition of cell wall in bacteria attribute to primary stain retention Gram Positive -contain a thick peptidoglycan with numerous teichoic acid cross-linkages

Thick teichoic acid cross-links contribute to the ability of Gram Positive bacteria to resist decolorization

Acid Fast Staining Designed for certain bacteria that contain cell walls containing long chain fatty (mycolic) acids that render the cells resistant by retaining the carbol fuchsin even with acid fast decolorizers Microorganisms that resists decolorization by an acid alcohol is called acid fast There are two main types of acid-fast stains:  Zhiel-Neelsen  Kinyoun’s All microorganisms are said to be nonacid fast except for Mycobacterium and Nocardia (Slightly acid fast)

Gram negative

Zhiel Neelsen method

Also known as the steam method Utilizes heat or steam to facilitate the penetration of the primary stain (Carbol Fuchsin) into the bacterial cell wall that is primarily filled with mycolic acid Thick mycolic acid is seemingly impenetrable without heat facilitation more so if stain other than carbol fuchsin is used.

Kinyoun’s method Also known as the cold method Does not utilize steaming for the penetration of carbol fuchsin Concentration and contact of carbol fuchsin to the smear is increased and wetting agents such as tertigol is added to allow the penetration of the primary stain to the bacterial cell wall...