PAL Video:HIV & AIDS Set [VHS]

This practical, hands-on video training programme is designed for pathologists and laboratory scientists who are beginning to use "in situ" polymerase chain reaction (PCR) - a diagnostic technique for infectious diseases and cancer. This video presents a step-by-step demonstration that shows viewers how to succesfully use the technique in their own laboratories. Part 1 of the video reviews key preparatory steps and includes information on the protease, fixative, hot start manoeuvre and reagents. Part 2 demostrates the actual techniques of "in situ" PCR and "in situ" hybridization. The third part of the video goes on to explain the basics of molecular biology and histology so that viewers can interpret "in situ" PCR findings.

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People's Century : 1952 Living Longer ; PBS ; Medical Science ; Disease

VHS

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Microcards: Review Cards for Medical Students

These flashcards will help medical students organize and recall medical microbiology information for course exams and USMLE Step 1. The cards are grouped into sections on gram-positive, gram-negative, and other bacteria; RNA and DNA viruses; fungi; protozoa; and helminths. Each section begins with charts summarizing key information on the group of microorganisms, followed by cards for each individual microbe, which include clinical presentation, pathobiology, diagnosis/treatment, quick facts, and a case study. Schematic illustrations show the morphology and pathogenesis of different microorganisms.

A companion Website provides 70 USMLE-style questions and answers.

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Clinical Microbiology Made Ridiculously Simple (Medmaster)

A brief, clear, thorough, and highly enjoyable approach to clinical microbiology, brimming with mnemonics, humor, summary charts and illustrations, from AIDS to "flesh-eating bacteria" to ebola, mad cow disease, hantavirus, anthrax, smallpox, botulism, etc. Excellent Board review.

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Gram Staining

A differential staining technique was introduced by Hans Christian Gram in 1884, which is now known as Gram Stain technique. The technique comprises of a primary stain (typically crystal violet), a mordant (Gram’s Iodine), a decoloriser (ethyl alcohol) and a counterstain (dil. carbol fuschin). This technique exploits fundamental physiological differences between gram positive bacteria and gram negative bacteria. Once stained by primary stained and fixed by a mordant Gram positive bacteria resists decolorisation by alcohol and remain violet at the end of staining. Gram negative bacteria gets decolourised and must be stained by a counterstain and appear pink in colour at the end of staining. Although the original method devised by Christian Gram comprised of Gentian violet, Lugol’s Iodine, absolute alcohol and Bismarck brown, there are various modifications of Gram stain in practice. These modifications include Jensen’s modification, Kopeloff & Beerman’s modification, Weigert’s modification and Preston & Morrell’s modification.

The procedure adopted in our institution is as follows:
The smear is covered with few drops of crystal violet solution and allowed to act for one minute. The slide is then washed with gentle stream of running tap water. The smear is then covered with few drops of Gram’s Iodine and allowed to act for a minute and then washed in tap water. The smear is then decolourised by alcohol by until no more violet colour comes off the slide. This process is completed within 30 seconds to prevent overdecolourisation. The slide is washed in water and counterstained using dilute carbol fuchsin for 30 seconds. The slide is then washed in water and dried with blotting paper and observed under oil immersion objective.

For more information and commonly asked questions, visit www.microrao.com/staining.htm

Photo of Gram stained smear showing gram positive cocci and gram negative bacilli


For a detailed description, visit my recent post here or type http://medimicro.blogspot.com/2008/10/gram-stain.html.

For PDf version, click here or type www.microrao.com/micronotes/pg/Gram stain.pdf

Ziehl Neelsen staining

This differential staining method was introduced by Ehrlich in 1882 and was subsequently modified by Ziehl and Neelsen independently. This staining method is useful in staining Mycobacteria in clinical specimens. Mycobacterial cell wall is made up of a waxy material (mycolic acid) that normally does not allow ordinary stains to enter the cell. The staining technique comprises of a primary stain, a decolouriser and a counterstain. The primary stain, which is typically concentrated (strong) carbol fuchsin is made by dissolving the dye basic fuchsin in phenol. Basic fuchsin dissolves better in phenol than in water. Heating the slide softens the waxy material of cell wall and phenolised dye readily enters the cell. Once stained by this method, these bacteria do not readily decolorize by weak mineral acids. Such bacteria are called acid fast bacteria. The non acid fast structures in the smear are then visualized by counterstaining with methylene blue solution. The acid fast bacilli appear pink in colour.

The procedure adopted in our institution is as follows:
The smear is flooded entirely with concentrated carbol fuchsin solution and heated using a spirit lamp from beneath. The heating should be intermittent and should not be intense to boil the solution or dry it completely. Typically, flaming must be stopped once fumes arise and allowed to cool. The solution is then poured off and washed in gentle stream of running tap water. The smear is then covered with few drops of 20% sulfuric acid and allowed to act for 1-2 minutes and then washed in tap water. The process of decolourisation may be repeated until the smear is faintly pink or almost colourless. The smear is then washed in water and counterstained with methylene blue solution and allowed to act for 30 seconds. The slide is then washed in water and dried with blotting paper and observed under oil immersion objective.

A positive sputum sample typically contain pink coloured, rod shaped bacteria that are slightly curved, sometimes branching, sometimes beaded in appearance, present singly or in small clumps against a blue background of pus cells and epithelial cells.

For more information and commonly asked questions, visit www.microrao.com/staining.htm

Photo of acid fast bacilli in sputum smear

Antibiotic susceptibility testing

The isolate to be tested should be obtained in pure culture and 3-5 identical colonies are picked up using a sterile straight wire and inoculated into nutient broth (or Mueller Hinton broth) and incubated at 37^oC for 2-4 hours or until sufficient turbidity is achieved. Alternatively, for testing the fastidious organisms such as Haemophilus spp., N. gonorrhoeae, and streptococci, and for testing staphylococci for methicillin resistance, a direct broth or saline suspension of isolated colonies on 18- to 24-hour agar plate can be made. Either way, the turbidity must be matched to that of 0.5 McFarland turbidity prior to testing. This is done by visually comparing the inoculum tube and the 0.5 McFarland standard against a card with a white background and contrasting black lines. The turbidity of the broth culture is adjusted with sterile broth to obtain a turbidity comparable to that of the 0.5 McFarland standard.This results in a suspension containing approximately 1 to 2 x 10⁸ CFU/ml for E.coli ATCC 25922.

Within fifteen minutes of standardization of inoculum, a sterile cotton swab is dipped inside the tube and squeezed against the sides of test tube to remove excess of fluid. The moist swab is rubbed on the entire surface of a dry Mueller Hinton agar. This procedure is repeated by streaking two more times, rotating the plate approximately 60^o each time so as to obtain an uniform lawn growth. Finally the circular rim of the plate must be swabbed.

The surface of the medium is allowed to dry and filter paper disks containing known antibiotic is placed on the surface and pressed gently to ensure proper contact. Once placed, the discs should not me moved or relocated since diffusion of antibiotic starts instantly. The interdisc distence should be 24 mm to prevent zone overlapping. Ordinarily, no more than 12 discs should be placed on one 150 mm plate or more than 5 discs on a 100 mm plate. Within fifteen minutes of application of discs, the plate is inverted and kept in incubator. The plates are incubated in ambient air or CO2, as required.



After 18-24 hours of incubation, the plates are removed from the incubator and observed for good growth and zones of inhibition around the discs. The diameter of zone of inhibition is measured to the nearest mm and read from the Kirby-Bauer Chart and is reported accordingly as susceptible, moderately susceptible or resistant.

For more information, visit www.microrao.com/micronotes/susceptibility.htm