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What is the primary objective of Direct Microscopic Count (DMC)?
What is the primary objective of Direct Microscopic Count (DMC)?
What is the function of Newman's stain in the preparation of a DMC slide?
What is the function of Newman's stain in the preparation of a DMC slide?
Which method would NOT be classified as a conventional method for detecting microbes in food?
Which method would NOT be classified as a conventional method for detecting microbes in food?
In the membrane filter method, what is the size of the pores used for filtering bacteria?
In the membrane filter method, what is the size of the pores used for filtering bacteria?
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What is the purpose of using a Petroff-Hausser chamber in microbial counting?
What is the purpose of using a Petroff-Hausser chamber in microbial counting?
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Which step is crucial to prevent cracking when preparing a DMC slide?
Which step is crucial to prevent cracking when preparing a DMC slide?
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Which of the following is NOT a direct counting method for assessing microbes?
Which of the following is NOT a direct counting method for assessing microbes?
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What is a common application of Direct Microscopic Count (DMC) in the food industry?
What is a common application of Direct Microscopic Count (DMC) in the food industry?
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What is the primary purpose of performing the pour plate method?
What is the primary purpose of performing the pour plate method?
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Which of the following is a benefit of the spread plate method?
Which of the following is a benefit of the spread plate method?
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What is a notable disadvantage of the spread plate method?
What is a notable disadvantage of the spread plate method?
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During the preparation of a 10-2 dilution from a solid food sample, what is the first step?
During the preparation of a 10-2 dilution from a solid food sample, what is the first step?
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How is the appropriate sample dilution generally prepared for the pour plate method?
How is the appropriate sample dilution generally prepared for the pour plate method?
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What is the main technique used in the Direct Epifluorescent Filter Technique (DEFT)?
What is the main technique used in the Direct Epifluorescent Filter Technique (DEFT)?
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Which method is most widely used for determining viable cells in food?
Which method is most widely used for determining viable cells in food?
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What is the first step in the membrane filtration process?
What is the first step in the membrane filtration process?
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What is a significant issue if there are fewer than 30 colonies on a plate?
What is a significant issue if there are fewer than 30 colonies on a plate?
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Why are membrane filters preferred for sterilizing sensitive materials?
Why are membrane filters preferred for sterilizing sensitive materials?
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For accurate results in the Standard Plate Count (SPC), what is the ideal range of colonies to count?
For accurate results in the Standard Plate Count (SPC), what is the ideal range of colonies to count?
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Which of the following is an advantage of using membrane filters?
Which of the following is an advantage of using membrane filters?
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Which factor does NOT affect the Standard Plate Count (SPC) method?
Which factor does NOT affect the Standard Plate Count (SPC) method?
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What is a common issue associated with counting colony-forming units if there are more than 300 colonies on a plate?
What is a common issue associated with counting colony-forming units if there are more than 300 colonies on a plate?
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What does the acronym DEFT stand for in the context of membrane filtration?
What does the acronym DEFT stand for in the context of membrane filtration?
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What is the purpose of using selective media in culture-based methods?
What is the purpose of using selective media in culture-based methods?
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Which process is crucial after filtering a sample through a membrane?
Which process is crucial after filtering a sample through a membrane?
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What outcome is expected by blending and homogenizing food samples before using the Standard Plate Count method?
What outcome is expected by blending and homogenizing food samples before using the Standard Plate Count method?
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In which of the following industries is membrane filtration particularly useful?
In which of the following industries is membrane filtration particularly useful?
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What will happen to viable cells when stained with fluorescent dyes in the membrane filtration process?
What will happen to viable cells when stained with fluorescent dyes in the membrane filtration process?
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What is done after rinsing the funnel with sterile buffered water?
What is done after rinsing the funnel with sterile buffered water?
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What is the primary purpose of tetrachloroethane in the staining process?
What is the primary purpose of tetrachloroethane in the staining process?
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Which component of the staining process fixes the smear?
Which component of the staining process fixes the smear?
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What is the advantage of using Direct Microscopic Count (DMC) for assessing milk quality?
What is the advantage of using Direct Microscopic Count (DMC) for assessing milk quality?
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What is a significant disadvantage of the DMC method?
What is a significant disadvantage of the DMC method?
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Which of the following correctly describes the process of direct counting on membrane filters?
Which of the following correctly describes the process of direct counting on membrane filters?
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Why is DMC not suitable for pasteurized milk?
Why is DMC not suitable for pasteurized milk?
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How is the density of microbial cells (DMC) per milliliter of milk calculated?
How is the density of microbial cells (DMC) per milliliter of milk calculated?
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What role does methylene blue play in the staining process?
What role does methylene blue play in the staining process?
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Study Notes
Direct Counting Methods
- Direct counting methods involve observing food samples directly or using filter paper to capture microorganisms.
- The captured microorganisms are then observed under a microscope.
- Direct Microscopic Count (DMC) involves directly observing microorganisms in food samples using a microscope.
- DMC involves making a smear of a food specimen or culture on a microscopic slide and staining with a dye.
- The cells are then counted using an oil immersion microscope at different magnifications.
- DMC is commonly used for raw milk and other dairy products.
- DMC assesses the microbial quality of the food product.
Direct Microscopic Count (DMC) Procedure
- Take 0.01 ml of milk with a sterile pipette and spread it evenly on a grease-free slide.
- Dry the smear on a warm surface at 40-45°C.
- Do not heat-fix the slide on a direct flame.
- Immerse the slide in Newman’s stain for up to 1 minute.
- Newman’s stain removes milk fat, fixes the smear, and stains the bacteria.
- The tetrachloroethane in the stain dissolves fat globules, ethyl alcohol fixes the smear, and methylene blue stains the smear.
Determining DMC
- Using oil immersion, determine the number of micro-organisms (individual cells or clumps) in a number of fields of the sample.
- Count the bacterial cells in random fields.
- The bacterial populations are high, so a significant number of fields are chosen for accurate counting.
- Calculate the average number of clumps per field and multiply by the microscopic factor to get the DMC per millilitre of milk.
- Interpret the results and compare with the standards to determine the quality of the milk sample.
Advantages of DMC
- Widely used to screen raw milk supplies for acceptable bacterial loads.
- Easy to perform.
- Requires less time.
- Large numbers of samples can be screened quickly
- Estimates the number and type of bacteria and somatic cells in milk samples.
- Used as a guide to identify bacterial types in milk samples.
- Determines the source of contamination in milk.
Disadvantages of DMC
- Not considered a fool-proof or legal method.
- Can cause eye strain for the operator.
- Not reliable because both viable and non-viable cells are counted.
- Not suitable for pasteurized milk.
- Requires experience to carry out.
- Results aren't reproducible because microorganisms are distributed unevenly.
Direct Counting on Membrane Filters (DCMF)
- Membrane filters with a pore size smaller than bacteria retain bacteria.
- The retained bacteria are then counted using a microscope.
- The procedure involves concentrating/collecting bacteria on polycarbonate filters by filtering a known volume of homogenized sample.
- Retained bacteria are stained and counted.
- Place the membrane on a nutrient agar media or absorbent pad saturated with the chosen culture media and incubate.
- Following growth, the colonies are counted.
DCMF Step-by-Step Procedure
- Collect the sample and make any necessary dilutions.
- Choose the appropriate nutrient or culture medium, dispense the broth into a sterile Petri dish, and saturate the absorbent pad.
- Flame the forceps and remove the membrane from the sterile package.
- Place the membrane filter into the funnel assembly.
- Flame the pouring lip of the sample container and pour the sample into the funnel.
- Turn on the vacuum and allow the sample to be completely drawn through the filter.
- Rinse the funnel with sterile buffered water and allow the liquid to be drawn through the filter.
- Flame the forceps and remove the membrane filter from the funnel.
- Place the membrane filter into the prepared Petri dish.
- Incubate at the proper temperature and for the appropriate time period.
- Count and confirm the colonies and report the results.
Uses of Membrane Filters
- They are used extensively to sterilize materials that can be damaged by heat sterilization.
- These include nutritional supplements for culture media and pharmaceutical products like drugs, hormones, sera, and vitamins.
- They are used to monitor drinking water and air quality.
- They are useful for bacterial monitoring in pharmaceutical, cosmetic, electronics, food, and beverage industries.
- Remove bacteriostatic or bactericidal agents that aren't removed using pour plate, spread plate, or MPN techniques.
- Can be used to allow selective passage of the desired organism by selecting a membrane filter of appropriate porosity.
Advantages of DCMF
- Well suited for samples containing low numbers of bacteria.
- Facilitates concentrating bacteria by filtering large sample volumes.
- Small food sample volumes can be used.
- Increasing the efficiency of the membrane filter method by staining with fluorescent dyes (e.g., acridine orange) and observing under an epifluorescence microscope (DEFT: Direct Epiflorescence Filter technique). Viable cells fluoresce green, and non-viable cells appear orange.
- Useful for enumerating microorganisms from a variety of foods.
Direct Epifluorescent Filter Technique (DEFT)
- Developed for rapid enumeration of microorganisms in raw milk samples.
- The method involves membrane filtration, capturing microorganisms, and staining with a fluorochrome, acridine orange.
- After staining, the membrane is mounted on a microscope slide and the stained microorganisms are visualized and counted with an epifluorescent microscope.
- The procedure takes as little as 30 min.
Culture-Based Methods
- Involve examining microorganisms in food by encouraging them to multiply in a liquid or solid medium.
- On solid agar media, bacteria develop as colonies, and counting these viable colonies determines the food's microbial load.
- Enumerating microorganisms using culture-based methods can be done using the Plate Count Method or Most Probable Number (MPN) technique.
Plate Count Methods
- Referred to as Total Plate Count (TPC), Standard Plate Count (SPC), or Aerobic Plate Count (APC).
- The most widely used conventional method for determining viable cells or colony-forming units (CFU) in foods.
- SPC involves homogenizing the sample, serially diluting it in an appropriate diluent, plating in or on suitable agar media, incubating at an appropriate temperature for a given time, and counting visible colonies as CFU.
Principle of the Plate Count Method
- Based on the principle that each viable bacterial cell multiplies and grows into a visible colony.
- Counting the number of colonies provides information about the bacterial cells present in the sample.
- Counts are determined by taking the average of replicate plates showing 30-300 colonies.
Factors Affecting SPC
- Sampling method.
- Distribution of microorganisms in food.
- Nature of the food microbes.
- Nutrient adequacy of the plating media.
- Incubation temperature and time.
- Type of diluents used.
- Presence of other competing organisms.
- Plating on selective media for specific organisms is limited by the degree of inhibition and effectiveness of selective/differential agents used.
SPC Variations
- Pour Plate Method: Appropriate dilution of the sample is mixed with agar medium, allowed to set, and incubated at a suitable temperature. Colonies developing on the surface and subsurface of the agar plate are counted. Proper mixing of sample with agar medium is necessary to get isolated colonies.
- Spread Plate Method (Surface Plating Method): Diluted sample is spread on the surface of a pre-poured, hardened agar plate using a glass rod. The plate is incubated at an appropriate temperature, and colonies growing on the surface are counted.
Advantages of the Pour Plate Method
- Suitable for heat-sensitive psychrotrophs as they don't come in contact with molten agar.
- Provides information on colony features useful in presumptive identification, especially on selective media.
- Favors strict aerobes on the surface, but microaerophils grow slowly.
Disadvantages of the Pour Plate Method
- Difficult to count colonies due to spreaders and crowding.
Advantages of the Spread Plate Method
- Suitable for heat-sensitive psychrotrophs in food as they do not come in contact with molten agar.
- Enables providing colony features useful in presumptive identification especially on selective media.
- Favors strict aerobes on surface, but microaerophils grow slowly.
Disadvantages of the Spread Plate Method
- Problem of spreaders and colony crowding makes the enumeration difficult.
Key Considerations for SPC
- Collect food samples randomly.
- Weigh 1 g of solid samples (vegetables/fruits) and homogenize in a pestle and mortar.
- Mix thoroughly with 9 ml of sterile diluent to make a 10-1 dilution.
- Transfer 1 ml of suspension from the 10-1 dilution to a 9 ml sterile blank using a sterile pipette to make a 10-2 dilution.
- Prepare dilutions up to 10-6.
- Liquid samples can be used directly in dilutions.
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Description
This quiz covers direct counting methods used in food microbiology, particularly focusing on Direct Microscopic Count (DMC) techniques. Learn how to evaluate microbial quality in food samples, specifically through steps involving the observation of microorganisms under a microscope. The quiz also delves into the procedure of preparing samples for microscopic examination.
