Dr. Shweta Mittal

Microbiology Researcher and Technologist


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COAGULASE TEST

Coagulase test is used to differentiate Staphylococcus aureus (positive) from Coagulase Negative Staphylococcus (CONS). Coagulase is an enzyme produced by S. aureus that converts (soluble) fibrinogen in plasma to (insoluble) fibrin.Staphylococcus aureus produces two forms of coagulase, bound and free.

  1. Slide coagulase test is done to detect bound coagulase or clumping factor.
  2. Tube coagulase test is done to detect free coagulase.

Slide Coagulase Test: Detects clumping factor (formerly referred as cell-bound coagulase)

  • Clumping factor directly coverts fibrinogen to fibrin causing agglutination
  • Heavy suspension of organism is made on glass slide and mixed with drop of plasma
  • Agglutination indicates a positive test:
  • ƒ IndicatesStaphylococcus aureus
  • ƒ Some species of Coagulase negative staphylococcus can be positive
  • Negative results should be confirmed

Slide Coagulase Test Procedure

  1. Emulsify a staphylococcal colony in a drop of water on a clean and grease free glass slide with a minimum of spreading. (If the isolate does not form a smooth, milky suspension, do not proceed with the test).
  2. Make similar suspensions of control positive and negative strains to confirm the proper reactivity of the plasma.
  3. Dip a flamed and cooled straight inoculating wire into the undiluted plasma at room temperature, withdraw, and stir the adhering traces of plasma (not a loopful) into the staphylococcal suspension on the slide. Flame the wire and repeat for the control suspensions.
  4. Read as positive a coarse clumping of cocci visible to the naked eye within 10 seconds. Read as negative the absence of clumping or any reaction taking more than 10 seconds to develop, but re-examine any slow reacting strains by the tube coagulase test.

Observation

  1. Coagulase Positive: Macroscopic clumping in 10 seconds or less in coagulated plasma drop and no clumping in saline or water drop.
  2. Coagulase Negative: No clumping in either drop

Interpretation: Slide coagulase test is the main method used to identify S. aureus in clinical laboratories but it has some limitations.

  1. About 15% of ordinary strains of S. aureus and many more of MRSA give negative reactions.
  2. Few species of coagulase negative staphylococci give positive reactions.

Note: All coagulase negative slides must be confirmed using tube coagulase test as the definitive test for S. aureus. 

Tube coagulase Test

  1. Detects staphylococcus which reacts with coagulase-reacting factor (CRF)
  2. CRF is a thrombin-like molecule
  3. Staphylo-coagulase and CRF combine to indirectly convert fibrinogen to fibrin
  4. A suspension of organism is suspended and incubated with plasma at 37°C
  5. Clot formation within 4 hours indicates a positive test
    • ƒ Positive test indicates Staphylococcus aureus
    • ƒ Some species of Coagulase negative staphylococcus can be positive.
    • NOTE: Negative tubes should be held overnight at room temp.
    •  Some species possess enzyme that can cause dissolution of clot after prolonged incubation

Tube Coagulase Test Procedure

  1. Prepare a 1-in-6 dilution of the plasma in saline (0.85% NaCl) and place 1 ml volumes of the diluted plasma in small tubes.
  2. Emulsify several isolated colonies of test organism in 1 ml of diluted rabbit plasma to give a milky suspension.
  3. Incubate tube at 35oC in ambient air or in water bath for 4 hours.
  4. Examine at 1, 2 and 4 hour for clot formation by tilting the tube through 90°. (Clots may liquefy after their formation)
  5. Leave negative tubes at room temperature overnight and re-examine.
    (This step is essential, for some strains of S. aureus, including many MRSA, produce a delayed clot which is rapidly lysed at 37°C by the organism’s staphylokinase.

Observation

Read as positive any degree of clot formation. Often the plasma is converted into stiff gel that remains in place when the tube is tilted or inverted, but sometimes clots are seen floating in the fluid.

Coagulase Test

  1. Coagulase Positive: Clot of any size eg. Staphylococcus aureus
  2. Coagulase Negative: No clot (plasma remains wholly liquid or shows only a flocculent or ropy precipitate). eg. Staphylococcus epidermidis

Note: Rabbit plasma is preferable, as it gives better clotting, is free from inhibitors and is safe. Human plasma contains sodium citrate as anticoagulant, and some citrate utilizing bacteria such as Enterococcus faecalis can destroy the anticoagulant and cause clotting. False positive or false negative results can occur if the plasma is not sterile.

 

 

 

 

 


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Oxidase Test

The oxidase test is used to identify bacteria that produce cytochrome c oxidase, an enzyme of the bacterial electron transport chain. When present, the cytochrome c oxidase oxidizes the reagent (tetramethyl-p-phenylenediamine) to (indophenols) purple color end product. When the enzyme is not present, the reagent remains reduced and is colorless.

All bacteria that are oxidase positive are aerobic, and can use oxygen as a terminal electron acceptor in respiration. This does NOT mean that they are strict aerobes. Bacteria that are oxidase-negative may be anaerobic, aerobic, or facultative; the oxidase negative result just means that these organisms do not have the cytochrome c oxidase that oxidizes the test reagent. They may respire using other oxidases in electron transport. Bacterial genera characterized as oxidase positive include Neisseria and Pseudomonas. Genera of the Enterobacteriaceae family are characterized as oxidase negative.

A positive oxidase result given by Pseudomonas aeruginosa (left disc) is indicated by a purple color. A negative oxidase result given by Escherichia coli (right disc) is indicated by the lack of color change. Both organisms (24 hrs old) were rubbed on a dry filter (Oxidase Disc-Himedia).

oxidase

Principle of the oxidase test reaction.

  1. Tetra-methyl-p-phenylenediamine dihydrochloride (TMPD), the oxidase reagent, is electron rich (reduced) and has no color.
  2. In bacteria that contain the enzyme cytochrome oxidase, one electron from each of four cytochrome c molecules will be temporarily transferred to the enzyme.
  3. This creates four electron-poor cytochrome c molecules and an electron-rich cytochrome oxidase enzyme.
  4. As the final step in respiration, the cytochrome oxidase enzyme transfers four electrons to molecular oxygen and along with four protons, forms two molecules of water, returning the cytochrome oxidase enzyme to its original state.
  5.   Instead of acquiring an electron from another component in the electron transport chain, an electron-rich TMPD molecule passes an electron to the electron-poor cytochrome c.  Cytochrome c returns to its original state and the resulting electron-poor (oxidized) TMPD


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Food Safety, adulteration and hygiene: A Global Issue

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Recently the Maggi issue was a word of every mouth. Findings says that Maggi was found adulterated with MSG (mono-sodium glutamate) and had higher content of lead, which is a very harmful if gets accumulated in human body. are we really concerned with the food safety issues or it was just a issue like various other political issues with catches fire and then eventually gets cool within a week. After this various reports were in news paper like insects were found in Glucon D in bulandsahar (U.P), 24% samples of milk were found adulterated in Maharashtra; Urea, coliform, E.coli, Salmonella were found in milk samples from Andhrapradesh; Center for Science and environment investigated transfats in Burger and Bhujiya. So, what is the action ? Nothing..what the concerned people are doing for the sake of consumer safety.

Major requirements for giving a solution to this matter is

1.) To appoint more food inspectors in every city for regular inspections of street vendors, small restaurants, MSME manufacturing units, big hotels, resorts and various other food establishments.

2.) Consultants should be appointed for providing guidelines to food safety and hygiene to these food establishments.

3.) Awareness among local people about the quality of what they are eating and drinking by organizing meetings and educating them about the ill effects of contaminated and adulterated food.

4.) Improving the hygiene standard in manufacturing facilities and food establishments using better technology.

5.) FSSAI should establish at least one lab in each city so as to analyse the sample load within proper time and deal with issues related to food adulteration and hygiene.


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Adultration of ayurvedic medicines

It is a strong belief in India that traditional medicines shall be used for treatment of both day to day and chronic ailments. All of us believe in and trust the medicines from the Ayurveda, Siddha, Unanai and Homeopathy systems. With the availability of superior technologies for testing the quality of Herbal medicines, the quality of these products has improved. But the dark side is these medicines are adulterated with modern medicines often at higher levels than recommended to achieve satisfactory results. Due to prolonged consumption of these adulterated medicines, patients may have serious to irreversible side effects.

Randomly I just checked few medicines my relatives were taking and I got shoking results. Those were positive for steroids adulteration.

If you or a family member is using an unlabeled, loose herbal medicine and want to get a lab test for steroids in Ayurvedic medicines or other herbal medicines you can contact any testing labs available in almost every city and get the sample tested to avoid long term side effects.

It’s a request, before taking the prescription you just get your sample (2-3 gm) tested. Below is a picture of steroid test. (1-blank, 2-std, 3,4,5 are samples). The color development in all three samples is comparable to standard. This concludes the presence of steroids in tested medicines.

Steroid Identification test

Steroid Identification test


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Quality concern of street foods

Major population of cities like Jaipur have their breakfast, snacks and dinner in street food shops. The shop vendors have very thin knowledge of food quality, hygiene and processing practices. This habit has negative aspects on the consumer’s health. Our studies showed that hygiene practices during sales, operations are not respected by all categories of vendors of food products. Also, it is observed that street food vendors sometimes sit close to the waste waters and garbage. in such cases, with poor conditions in hot summers and rainy season, food gets contaminated with microorganisms under favorable conditions. Current reports gives a idea of what level of pathogenic organism we are having in food. As a result of which there is a compromise in microbial quality of food. the food which are half cooked, uncooked or not recooked at at highest risk of contamination from various Salmonella sp., Shigella sp., E. coli, Vibrio sp. It probably makes a high alert for consumer and vendor to not to compromise with food quality onto which large population is dependent. In our study, food items like paani puri, vegetable poha, aaloo tikki were sampled from streets of jaipur, Rajasthan. Various pathogens like S. aureus, Vibrio sp. , E. coli were found in these samples. Vendors need to be take care for quality concern.