FUNGI TAXONOMY , MORPHOLOGY AND CLASSIFICATION

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FUNGI TAXONOMY, MORPHOLOGY & CLASSIFICATION

fungi media

fungi media 

fungi spore dispersol

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fungi spore dispersol.ppt

PRESERVATIVE EFFICACY TEST

 

Preservative Efficacy Testing (PET)

Preservative Efficacy Testing (PET) evaluates the effectiveness of a cosmetic or over-the-counter (OTC) pharmaceutical product’s preservative system, which is designed to inhibit the growth of microorganisms that could be introduced during manufacturing or by users during regular use.

By simulating these conditions, Preservative Efficacy Testing helps determine if products are capable of remaining free from harmful microbes, safeguarding consumer health and maintaining product integrity.

This testing is a critical safety and quality assurance measure for manufacturers in the health and beauty industries.

PRINCIPLE OF TEST:

Antimicrobial preservatives are added to products to prevent or limit microbial contamination, which can occur during normal conditions of storage and use.  The efficacy of an antimicrobial preservative may be enhanced or diminished by the active constituent of the preparation, or by the formulation in which it is incorporated, or by the container and/or closure being used as the final packaging material.

The test method must be qualified for the product under evaluation to ensure the correct diluent is used in assays for surviving microorganisms.

The product is inoculated with specified number of each challenge organism.  The inoculated product is held at room temperature for 28 days.  It is examined by the duplicate plate count method to determine the number of viable microorganisms which survive at each specified time interval.

A preservative efficacy test (PET), also known as an antimicrobial effectiveness test (AET) or challenge test, is a crucial procedure used to evaluate the ability of a product's preservative system to prevent the growth of microorganisms that may be introduced during manufacturing or consumer use.

The goal of a PET is to ensure that a product remains safe for its intended shelf life by effectively controlling microbial contamination. This is particularly important for multi-dose products (e.g., lotions, creams, eye drops) where repeated opening and use can introduce microorganisms.

Here is a general overview of a typical preservative efficacy test protocol:

1. Preparation of Test Materials

·         Test Sample: The product to be tested is divided into separate containers, one for each test microorganism. It is often recommended to use the final container closure system to simulate real-world conditions.

·         Test Microorganisms: A panel of standard microorganisms is used to represent common contaminants. The most common strains include:

o    Pseudomonas aeruginosa (ATCC 9027)

o    Staphylococcus aureus (ATCC 6538)

o    Escherichia coli (ATCC 8739)

o    Candida albicans (ATCC 10231)

o    Aspergillus brasiliensis (ATCC 16404)

o    Note: Other relevant environmental isolates from the manufacturing facility may also be included.

·         Preparation of Inoculum: The microorganisms are grown under specific conditions to a desired concentration, typically to produce a suspension of about 1×108 colony-forming units (CFU)/mL.

2. Inoculation and Incubation

·         Inoculation: A small volume (e.g., 0.5% to 1.0% of the product volume) of the prepared microbial suspension is added to each container of the test product. The final concentration of microorganisms in the product should be between 1×105 and 1×106 CFU/mL.

·         Mixing: The inoculated product is thoroughly mixed to ensure a homogeneous distribution of the microorganisms.

·         Incubation: The inoculated samples are incubated at a prescribed temperature, typically in the range of 20−25∘C or 22.5±2.5∘C, for a period of at least 28 days.

3. Sampling and Microbial Enumeration

·         Sampling Intervals: Samples are withdrawn from each container at specific time intervals to determine the number of surviving microorganisms. Standard time points often include:

o    Initial count (time 0)

o    Day 7

o    Day 14

o    Day 28

o    Some protocols may include additional time points, such as Day 2, Day 21, or re-challenge on a later date.

·         Neutralization: A crucial step is to neutralize the antimicrobial activity of the product's preservative system before plating the samples. This ensures that the microorganisms are not killed on the agar medium, allowing for accurate enumeration of the surviving population. Neutralizers such as polysorbate 80 and lecithin are commonly used.

·         Plate Counting: The neutralized samples are serially diluted and plated onto appropriate agar media. The plates are then incubated, and the colonies are counted. This process allows for the calculation of the number of viable microorganisms per gram or milliliter of the product at each time interval.

4. Calculation and Acceptance Criteria

·         Log Reduction Calculation: The log reduction is calculated by comparing the initial microbial count to the count at each sampling interval. The formula is as follows:

o    Log Reduction=log10​(Initial Count)−log10​(Count at Time t)

·         Acceptance Criteria: The product is deemed adequately preserved if the microbial counts meet the specific criteria outlined in the relevant pharmacopoeia or standard. The criteria vary depending on the product category (e.g., injections, topical products, oral preparations).

o    For example (simplified criteria):

§  Bacteria: A certain log reduction (e.g., 1 or 2 log) is required at Day 7, followed by a further reduction or no increase at Day 14 and Day 28.

§  Yeast and Mold: The count should not increase from the initial count, or a specified log reduction may be required.

Important Considerations

·         Suitability Testing: Before the official test, a "suitability of recovery" or "neutralizer validation" test is performed. This confirms that the chosen neutralization method effectively inactivates the preservative without harming the test microorganisms, ensuring accurate enumeration.

·         Environmental Isolates: Incorporating microorganisms isolated from the manufacturing environment can provide a more robust test, as these strains may have a higher resistance to the preservatives used.

·         Product-Specific Factors: The effectiveness of a preservative can be influenced by the product's pH, the presence of other ingredients, and the packaging. A thorough PET should account for these factors.

·         Shelf-Life and Stability: PET is often performed as part of a product's stability study to ensure the preservative system remains effective over its entire shelf life.

 

RESAZURIN REDUCTION TEST

 

Resazurin Reduction Test (RRT)

Resazurin reduction t is another method of dye reduction test and the principle of this test is nearly similar to methylene blue reduction test. In MBRT the time for reduction of the dye is measured, while in RRT, at a fixed period time, specific shade of colour and its intensity is measured.  There are two variations in RRT of testing milk. One is 10 min RRT test that can be used as a rapid platform test for quick assessment of milk at the raw milk reception dock. The other one is a one hour RRT performed in the lab. In this we will talk about Rapid RRT.

Principle                 

Unlike methylene blue the resazurin undergoes reduction through a series of colour shades viz., blue, purple, and lavender, pink before completely getting reduced to colourless.

Resazurin dye which is blue in colour at the oxidation-reduction potential of + 0.3 volts undergoes an irreversible change to pink colour compound (resorufin) when the redox potential reduces to +0.2 volts.

When the redox potential is reduced further to + 0.1 volts or less, the colour of dye changes to colourless (dihydroresorufin), which is a reversible reaction. Usually, the degree of reduction of the dye is measured after a fixed time of incubation of milk sample in the presence of dye.

The reduction of dye to a particular shade of colour is dependent upon the extent of depletion of oxygen by metabolic activity of microbes. The colour change is measured with the help of a Lovibond colour comparator and a standard resazurin disc.

Standard solution of resazurin

·        One tablet of Resazurin is dissolved in 50 ml of cold sterile glass distilled water by gentle heating to facilitate the dissolving. This is the bench solution for direct use and should always be used as fresh.

·        Alternatively dissolve 0.05 g of resazurin powder in 100 ml of distilled water and boil the contents for 30 min. This will make a standard solution of 0.05%, which should always be kept in a cool and dark place, stored in an amber coloured bottle.

·        The bench solution (0.005%) for regular use should be prepared freshly by diluting the standard solution with distilled water i.e. 1 ml of standard solution with 10 ml of distilled water.

 

Procedure

1.      Take 10 ml of milk into a test tube and add 1 ml of working solution of Resazurin solution.

2.      Put air tight closure to prevent oxygen entry

3.      Invert the test tubes to mix the milk and Resazurin solution.

4.      Place the test tubes in a thermostatically maintained water bath at 37 ±5 °C and note down the time of incubation (10 min).

5.      At the end of incubation match the colour of the milk with one of the colour standards of Resazurin disc.

Resazurin chemical structures 

Grading of milk

The quality of raw milk is adjusted by using the following parameters.

 

Advantages of dye reduction test

Used for estimating the suitability of milk for liquid consumption.

These tests are cheaper and less time is required.

With the help of these tests the activity is measured rather than the number of bacteria.

Unlike the artificial media used in SPC, in milk the natural environment for microbes is present.

In case of RRT, the results can be measured in a shorter time.

Some of the bacteria capable of reducing the dye may not develop colonies on the medium used in SPC.

Disadvantages

Rate of reduction of dye varies considerably and is related to species and the rate at which different micro-organisms grow at a particular temperature.

Inhibitory substances like penicillin and other antibiotics prevent the growth of bacteria and thus increase the reduction time.

Not suitable for classifying milk with low bacterial counts of less than 10⁵ /ml.

Reduction capability may vary because of variation in proportion of bacteria carried into cream layer by the rising fat globule.

These tests do not give indication for the type of micro-organisms present.

Temperature of incubation used during these tests is not the optimum for majority of the micro-organisms present in milk.

Not suitable for testing quality of pasteurized milk intended for processing because of the low number of micro-organisms.

Require continuous attention until reduction takes place. 

FUNGI SPORES DISPERSAL

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FUNGI SPORE DISPERSAL

FUNGI SPORE DISPERSAL

Reduction in sexuality in fungi

Reduction in sexuality in fungi https://docs.google.com/presentation/d/1OO-5YuLKydLbBgvriEzm2PY2jSVO0Fhp/edit?usp=drive_link&ouid=110279467536365899548&rtpof=true&sd=true

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