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Detection of Microbe
Traditional methods
| Question | Answer |
|---|---|
| What is/are the essential part of any quality control or food safety plan? | detection and enumeration of microorganisms in food |
| Time consuming method of detecting foodborne pathogenic bacteria because of the need for growth in culture medium, isoltion, biochem, & others | Traditional methods |
| Why are traditional methods of detecting foodborne pathogenic bacteria are often time consuming ? | the need for growth in culture media, followed by isolation, biochemical and/or serological identification, and in some cases, subspecific characterization |
| Advances in technology have made detection and identification ------, more ------, more -------, and more ----- than traditional assays | faster, sensitive, specific, convenient |
| used to detect the presence of pathogens in raw and processed foods immediately | Rapid detection methods |
| Rapid methods are also------ enough to detect pathogens that are present in low numbers in food. | sensitive |
| basic to food microbiology | examination of foods for the presence, types, and numbers of microorganisms and/or their products |
| every method has certain ----- ------ associated with its use | inherent limitations |
| Method for viable cells | Standard plate counts (SPC) |
| Method for statistical determination of viable cells | The most probable numbers (MPN) method |
| Method to estimate numbers of viable cells that possess reducing capacities | Dye reduction techniques |
| Method for both viable and non-viable cells | Direct microscopic counts (DMC) |
| What method involves the homogenization of food samples by blender, followed by serial dilution, plating in agar medium, incubation, and counting by Qubec or electronic counter? | Conventional SPC method |
| When total viable counts are reported for a product, the counts should be viewed as? | a function of at least some of the following factors: sampling methods are employed, nature of food biota, type of diluent, etc. |
| description of Standard Plate Counts (SPC) for viable cells | Most widely used for determining numbers of viable cells or colony forming units (cfu) in food products |
| Surface plating offers advantages in determining the numbers of | heat-sensitive psychrotrophs |
| surface plating favors | strict aerobes |
| Why does Surface plating offers advantages in determining the numbers of heat-sensitive psychrotrophs in a food product | the organisms do not come in contact with the melted agar |
| in surface plating microaerophilic organisms tend to grow | slower |
| surface plating method aka | spread plate/plating method |
| Process of spread plating | 0.1ml of sample is pipetted onto surface of agar, then evenly spread by an l rod, incubated, and counte.d |
| Pour plating method process | 1ml sample pipetted in sterile plates, then sterile medium/agar is added and mixed with inococulum, incubated, then counted |
| Disadvantages of surface plating | problem of spreaders and crowding of colonies |
| In 1970s what type of blender was used for homogenization of samples? | Waring type blender |
| In 1971 what was used for homogenization of samples? | Colwell Stomacher (Sharpe and Jacson in England) |
| What device homogenizes specimens in a special plastic bag by the vigorous pounding of two paddles. | Stomacher |
| Why is stomacher preferred over blender? | No need to clean and store, no heat bildup during normal operational times, homogenates can be stored in stomacher bags in freezer, noise level is reduced, and it is less lethal than a blender |
| Normal operational times of stomacher | 2 minutes |
| Stomacher was shown to be less lethal than a blender to ------, Enterococcus faecalis, and Escherichia coli | Staphylococcus aureus |
| Stomacher was shown to be less lethal than a blender to Staphylococcus aureus, ------, and Escherichia coli | Enterococcus faecalis |
| Stomacher was shown to be less lethal than a blender to Staphylococcus aureus, Enterococcus faecalis, and-------- | Escherichia coli |
| a mechanical device that distributes the liquid inoculum on the surface of a rotating plate containing a suitable poured and hardened agar medium. | SPIRAL PLATER |
| In spiral plater, The dispensing arm moves from the near center of the plate toward the outside, depositing the sample in an ----- | Archimedes spiral. |
| In spiral plater, syringe disposes a continuously ---- volume of sample | decreasing |
| sample concentration that spiral plater disposes in single plate | 10,000:1 |
| colony development of spiral plater reveals higher density of desposited cells near the? | center of the plae |
| colony development of spiral plater reveals fewer density of desposited cells toward the? | edge |
| The enumeration of colonies on plates prepared with a spiral plater is achieved by use of a | special counting grid |
| Advantages of spiral plater: | fewer plates, dilution blanks, and pipettes required; 3-4 times more samples per hour can be examined; 50-50 plates can be prepared/hr; little trianing required for operation |
| Disadvantage of spiral plater | food particles may cause in blocking the dispensing stylus. |
| spiral plater is more suited for use with------foods such as milk | liquid |
| Pore size of membrane filters that will retain bacteria | 0.45um |
| Process of membrane filter | collect bacteria by filtering in given volume of diluent; place membrane in agar plate/absorbent pad; incubate; colony count |
| Method suited for samples that contain low numbers of bacteria | Membrane filter |
| DMC- direct microscopic count can be improved with what dyes? | fluorescent dyes |
| Count for the total dead and live cells in a special microscopic slide containing a premeasured grid | DMC/Direct cell method |
| counting chamber used in dairy industry | petroff-hausser counting chamber |
| filters that were among the earliest used | cellulose filters |
| filters that offer advantage of retaining all bacteria on top of the filter | nucleopore filters |
| DEFT meaning | Direct epifluorescent filter technique |
| method that allows for the direct microscopic determination of cells | DEFT |
| is a variation that allows one to determine viable cells only | Microcolony-DEFT |
| Dye used in microcolony-DEFT | acridine orange: fluorescent dye (fluorochrome) |
| HGMF meaning | Hydrophobic Grid Membrane Filter |
| Hydrophobic Grid Membrane Filter (HGMF) | The method employs a specially constructed filter that consists of 1,600 wax grids on a single membrane filter that restricts growth and colony size to individual grids. |
| The HGMF method can detect as few as --- cells per gram, and results can be achieved in 24 hours or so | 10 |
| HGMF can be used to enumerate all cfus or specific groups such as indicator organisms, fungi, -------- and ------. | salmonellae and pseudomonads |
| HGMF was given the ------ aproval for total coliforms, fecal coliforms, salmonellae, and yeasts and molds. | Association of Official Agriciltural Chemist (AOAC) |
| meaning of AOAC | Association of Official Agriciltural Chemist |
| The HGMF method allows the filtering of up to ---- g of food per membrane. | 1 |
| MCCM meaning | Microscopic Colony Count |
| involve the counting of microcolonies that develop in agar layered over microscope slides | Microscopic Colony Count (MCCM) |
| First process of MCCM by Frost | spreading of 0.1ml of milk-agar mixture over a 4cm2 area on glass slide, incubattion, drying &staining, and microcolony count |
| 2nd method of process for MCCM | 2ml of melted agar mixed with 2ml wared milk, spread 0.1 ml of agar over 4cm2 area, stain with thionin blue, and viewing |
| what stain can be sued for microscopic colony count? | thionin blue |
| The method was about three times faster, and 24-hour incubations gave counts equal to those obtained after 48 hours by the conventional plate count. | Agar droplets |
| advantages of agar droplets | dilution blanks not required, and only one petri dish per sample is needed |
| The method can be used with nonselective ingredients to make aerobic plate counts (APCs); an acceptable alternative to SPC, approved by AOAC | Dry film and related methods |
| A rehydratable dry film method consisting of | two plastic films attached together on one side and coated with culture media ingredients and a cold-water-soluble jelling agent |
| process of dry film and related methods | first 1ml of diluent is placed between two films; spread over nutrient area by pressing; incubate; observe |
| in dry film and related methods what is the color of microcolony on the nonselective film and why? | red; because of the presence of a tetrazolium dye in the nutrient phase |
| Plating method that don't use agar as solidifying agent; but is employed by inoculating sterile ingredients with food homogenates by mixing and holding to allow solidfication | redigel plating |
| culture method based on activity of several enzymes common to many food born organisms; special plates with hole/wells; do not allow characterization of colony features | sim plating |
| two plating methods under dry film? | redigel plating and sim plating |
| in redigel plating holding and solidification occurs in about how many minutes? | 30 minutes |
| benefits of dry film plates | save materials, equipment, low labor costs, no need to prep agar, etc. less storage, reduce biohazard and pastic waste |
| --------- make colonies countable at earlier stage giving faster results e.g. 24 hours instead of 3 days or 48-72 hours instead of 5-7 days depending on type of test | indicator dyes in dry film methods |
| what give clear colony identification | highly specific chromogens |
| disadvantages of dry film methods | plating issues still remain esp if sample has high bacterial load; odd organisms with unusual characteristics; Very high or very low pH products will require an adjustment to neutral pH |
| It is statistical technique to determine number of organisms in sample. | Most probable numbers |
| Most probable numbers 3 steps | presumptive test, confirmatory test, and completed test |
| In MPN, what 3 things are observed to determine microorganisms. | Turbidity, gas production and acid production |
| The method is statistical in nature, and MPN results are generally higher/lower than SPC results | higher |
| What organisms are assessed in the medium: ec broth | faecal coliform |
| What organisms are assessed in the medium: glucose azide minerals modified glutamate medium | faecal streptococci |
| What organisms are assessed in the medium: baird-parker broth | staphylococcus aureus |
| What three mpn medium are used to assess coliforms? | Lauryl sulfate tryptose broth, macconkey purple broth, and minerals modified glutamate medium |
| Advantages of MPN | relatively simple; specific group of mcirobes are determined by using selective/defferential media; method of choice for fecal coliform densities |
| disadvantage of MPN | use large colume of glasswares; lack of opportunity to observes microscopy, lack of precision |
| Two dyes employed in dye reduciton technique | methylene blue and resazurin |
| general process for dye reduction technique | supernatants of food added to standard solutions of dye; color change is observed |
| high number of organisms will involved less/more time for dye reaction to occur | less time |
| commonly used to determine number of viable organisms in raw milk | Methylene blue reduction test |
| What dye is used in methylene blue reduction test? | methylene blue |
| color change in methylene blue reduction test is from--- to--- | blue to colorless |
| color change in resazurin reduction test is from--- to--- | blue to pink |
| methylene blue (MB) is changes to | leucomethylene blue (lmb) |
| It is an example of rapid dye reduction test use to determine number of viable organism in food such as raw milk | Resazurin reduction test (rapid test) |
| In resazurin reduction test, resazurin is reduced to | resofurin |
| tiin resazurin reduction test results are obtained within--minutes | 10 minutes |
| Dye-reduction tests have a long history of use in the ----- industry for assessing the overall microbial quality of raw milk. | dairy |
| advantages of dye reduction: | ◼ are that they are simple, rapid, and inexpensive; ◼ only viable cells actively reduce the dyes. |
| disadvantages of dye reduction:: | -not all organisms reduce the dyes equally, and; they are not applicable to food specimens that contain reductive enzymes unless special steps are employed. |
| It has been found to be an excellent method for enumerating fastidious anaerobe | Roll tubes |
| gas that is added in screw capped tubed for roll tube technique to exclude oxygen | carbon dioxide and di hydrogen (h2+ co2) |
| are most widely used in the dairy industry for assessing the microbial quality of raw milk and other dairy products, and | DMCs |
| counitng methods developed by R.S. Breed | Breed count |
| In direct microscopic count liquid food sample are directly smeared, wehreas solid foods are diluted. How are fatty foods defattted? | fatty foods can be defatted using xylene or acetone |
| What liquid material can remove xylene and acetone after da=efatting of fatting foods for preparation of smear? | ethanol |
| In breed count method how many ml of sample is spread over 1cm2 area on slide? | 0.01ml |
| In breed count method, what do you call the slide used for counting and viewing? | Hemcytometer |
| what areas in hemacytometer are NOT counted? | lower and right sides |
| what areas in hemacytometer are counted? | Cells touching any of the three lines on the upper and left sides. |
| formula of bacterial count in breed method | Bacterial count= (total count/no. of squares) x (1/square volume) x dilution |
| two methods in direct microscopic counts: | breed count and howard mold counts |
| who developed howard mold counts method? | BJ Howard |
| Why did Howard mold count develop microscope slide method for? | for the purpose of monitoring tomato products |
| The method requires the use of a special chamber (slide) designed to enumerate mold mycelia; | Howard Mold Counts |
| Similar to the Howard mold count is a method for quantifying what bacterium in canned beverages and fruits | Geotrichum candidum |
| 3 common methods for surface assessment in food: | 1. Swab/swab-rinse method 2. Contact plate 3. Agar syringe/ Agar Sausage method |
| Other surfaces methods are: | ◼ Direct surface ◼ Sticky film ◼ Swab/agar slant ◼ Spray gun |
| ◼ It is the oldest and most widely used method ◼ Not only in the food and dairy industries but also in hospitals and restaurants | Swab/swab-rinse method |
| ◼ The replicate organism direct agar contact (RODAC) method employs special Petri plates poured with enough medium, resulting in a raised agar surface. ◼ When the plate is inverted, the hardened agar makes direct contact with the surface. | Contact plate |
| By this method, a 100-mL syringe is modified by removing the needle end to create a hollow cylinder that is filled with agar. | Agar syringe/ Agar Sausage method |
| melted agar is poured onto surface/utensils and the mold is plates on petri dish, ie., c. sporogenesis ensospores on stainless steel surfaces | Direct surface |
| pressing sticky film/tape against surface and pressing it on agar plate; i.e meat surfaces | Sticky film |
| sampling with cotton swabs transferred directly to slants | Swab/agar slant |
| based on impingement of a spray of washing solution against a cricumsbried area of surface and plating of the washing solution. more effective than swab method in meat surfaces test | Spray gun |
| Type of swab that – when used, the organisms must be dislodge from the fibers | Cotton swab |
| Type of swab that –when used, the organisms be released in the diluent upon dissolution of the alginates by sodium hexametaphosphate | Calcium alginates |
| n in the swab-rinse method presented by Koller how many seconds is the surface swabbed over 3cm2 area | 15 seconds |
| In contact plate how many ml of agar is poured to make raised agar surface? | 15.5-16.5 ml |
| In contact plate technique, When surfaces are examined that have been cleaned with certain detergents, it is necessary to include a neutralizer like what in the medium. | lecithin or tween 80 |
| disadvantage of contact plate | The covering of the agar surface by spreading colonies and its ineffectiveness for heavily contaminated surfaces. |
| method that uses plastic tubing; has been used largely by European workers for assessing the surfaces of meat carcasses, as well as for food plant surface | agar suasage |
| surfaces small in size/removable are placed in container immersed in diluent; energy generated makes the release of microorganisms into diluent. | ultrasonic devices |
| 3 sampling methods: | visual inspection, final rinse sampling, and swabbing |
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honeybunch
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