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6Microbiological testing
This is a mind map about food microbiology-6 microbial testing, including microbial quantity determination, sample collection and processing, Bioanalysis and related technologies, etc.
Edited at 2024-01-20 17:23:40- bacteria
This is a mind map about bacteria, and its main contents include: overview, morphology, types, structure, reproduction, distribution, application, and expansion. The summary is comprehensive and meticulous, suitable as review materials.
- Plant asexual reproduction
This is a mind map about plant asexual reproduction, and its main contents include: concept, spore reproduction, vegetative reproduction, tissue culture, and buds. The summary is comprehensive and meticulous, suitable as review materials.
- Reproductive development of animals
This is a mind map about the reproductive development of animals, and its main contents include: insects, frogs, birds, sexual reproduction, and asexual reproduction. The summary is comprehensive and meticulous, suitable as review materials.
6Microbiological testing
- bacteria
This is a mind map about bacteria, and its main contents include: overview, morphology, types, structure, reproduction, distribution, application, and expansion. The summary is comprehensive and meticulous, suitable as review materials.
- Plant asexual reproduction
This is a mind map about plant asexual reproduction, and its main contents include: concept, spore reproduction, vegetative reproduction, tissue culture, and buds. The summary is comprehensive and meticulous, suitable as review materials.
- Reproductive development of animals
This is a mind map about the reproductive development of animals, and its main contents include: insects, frogs, birds, sexual reproduction, and asexual reproduction. The summary is comprehensive and meticulous, suitable as review materials.
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- Outline
Microorganisms in food and their Detection of metabolites
Microbial count determination
standard plate count SPC
experiment process
sampling
dilution
ten times dilution
Inoculation: 2-3 serial dilutions, parallel controls
pouring method
Coating method
Detects heat-sensitive bacteria
More suitable for strictly aerobic bacteria
The colony morphology is obvious and the group characteristics can be observed.
Not to be confused with food particles
Colonies tend to overlap and mix
nourish
medium, conditions
Inverted culture: medium weight loss less than 15%
Prevent water droplets from dripping
Prevent the growth of bacteria
colony count
Select a plate with appropriate bacterial colony count, 30 to 300 CFU for bacteria, and 10 to 150 CFU for mold/yeast.
Methods: naked eye observation, colony counter
Identify colonies: Colonies are too small or similar to sample particles - dye reduction method, TTC (red tetrazolium, chromogenic medium), bacterial characteristics
calculate
Report, two significant figures, blank control has colony test results invalid
significance
Determine the degree of microbial contamination and hygiene quality
Predict food shelf life
reflect freshness
Monitor growth and reproduction dynamics
Biological product testing: contamination, probiotics
Other methods
Hydrated dry film method-culture medium
Principle: Prefabricated culture medium system, renewable hydration dry film, culture medium, cold water soluble gel, indicator, printed grid
Operation steps: dilution, inoculation, culture, counting, reporting
advantage
high productivity
Small error
Internationalization: Approval from official bodies
Fast: 6-14 hours can estimate accurate results within 24 hours 3-5 days to confirm mold and yeast counts
identification medium
rotation inoculation method
According to the Archimedean spiral as the inoculation trajectory, inoculate the sample at a decreasing rate
Automatic spiral inoculation, counting system
Counting method: There are more colonies in the middle and less around them. Calculate the number of colonies per unit area.
advantage
The measurement results are highly correlated with SPC
shortcoming
Nearest number determination method MPN
Utilize the special physiological functions of the microorganisms to be tested and use selective culture media (to reduce interference) to determine the presence and abundance of microorganisms.
Detection of microorganisms with nutritional and metabolic properties such as coliforms
easy to use
Microorganisms that are not dominant in the microbial community can be measured
coliforms
Gut bacteria, linked to fecal contamination
Aerobic and facultative anaerobic negative non-saccharomyces bacteria that ferment lactose to produce acid and gas under certain culture conditions.
Primary fermentation: LST, secondary fermentation BGLB for gas producers
The significance of detecting E. coli
Fecal contamination indicator bacteria to evaluate food hygiene quality
Evaluate the potential for contamination by enteric pathogenic bacteria, such as Salmonella, Shiga
Advantages as indicator bacteria
Many sources, large quantities, high detection rate
The external survival time is basically the same
Resistance to fungicides is basically the same
Easy to operate, no complicated equipment required
high sensitivity
Dye reduction counting method (estimation method)
Principle: Living cells contain reductase enzymes that can reduce specific fuels from one color to another.
Commonly used reagents
Methylene blue
blue to white
Resazurin
Dark blue turns to pink, white
Tetrazole
Colorless to red
Reduction time is inversely proportional to microbial concentration and is often used for milk hygiene and sperm activity.
advantage
Easy, fast and economical
shortcoming
The reducing ability of microorganisms is different and difficult to estimate. It is not suitable for foods containing reductases.
Direct microscopic counting method DMC
counting board direct counting
Petrovholzer cell counting chamber (general bacteria), hemocytometer (yeast/mold spores)
Count the number of microorganisms in a certain volume, but cannot distinguish miscellaneous bacteria
advantage
Fast, convenient, cell morphology analysis, slides easy to save
shortcoming
Only suitable for samples with high bacterial content, poor accuracy and easy to fatigue.
Other methods
Cotton swab smear method, sampling method
Detection of surface microorganisms
Applied to food, equipment surfaces, public places
Membrane filtration method, sample concentration
Samples containing very little bacteria can enrich the bacteria and improve the detection rate.
Not limited by sample volume
Detection method: SPC, microscopic counting, combined with staining method
Physical, chemical, molecular and immunological methods
physics
Impedance measurement
impedance
In biological materials with resistance, inductance and capacitance, the impedance that hinders the measurement of current is called impedance.
Principle: Microorganisms change the electrical properties of the culture medium, and the electrically inert substrate decomposes into electroactive molecules and ions. The conductivity of the culture medium increases and the impedance decreases.
Changes in conductivity over time
Detection time: the time from culture to the sudden change in impedance value, inversely proportional to the original bacterial count
Deduced the original bacterial count of microorganisms
Microorganisms have different impedance curves
Microbial identification
direct impedance method
The culture medium is put into a special measuring tube. After inoculation with microorganisms, electrodes are inserted into the culture medium to directly measure changes in the electrical properties of the culture medium.
key factors, culture medium
The tested bacteria➕produce monitorable impedance changes
indirect impedance method
Microbial metabolism produces carbon dioxide, etc., to reflect the metabolic activity of microorganisms
Carbon dioxide enters the test tube and reacts with potassium hydroxide to produce carbonate, which reduces its conductivity and records the change in conductivity.
advantage
No need to optimize the culture medium (can react without electricity)
Can measure media containing high salt content
Measurable microorganisms that produce very little or no measurable change in conductivity, such as yeast
The measurable food itself may interfere with the impedance measurement or even damage the electrodes.
Microcalorimetry
Chemical
Determination of ATP
Energy source for living cells, disappears two hours after cell death
Cellular ATP is constant, and its total amount is proportional to the number of bacteria, which can be calculated
The ATP content of prokaryotic exponentially growing cells is usually 2-6 nmol/mg dry weight.
Accurate measurement of ATP in cells of the same species
Common methods —Measurement of ATP by luciferase system
ATP participates in the luciferin-luciferase system, causing luciferin to emit light, and the amount of light emitted is proportional to ATP
Calculate cell number based on luminescence intensity
It can automatically measure and detect quickly. The results are consistent with the SPC method, saving time.
application
Determination of bacteria count in fermentation broth
Urine sample testing
Surface microbiology assay
Radiometry
Principle: Bacteria produce carbon dioxide when they metabolize carbohydrates, label trace elements into glucose or other sugar molecules, and detect the released carbon dioxide
The amount of radiation is proportional to the number of bacteria
Detection time is inversely proportional to the number of microorganisms
radioactive energy counter
application
Check for stomach problems by blowing air
Food microbiology testing
Fingerprinting and identification of food microorganisms
Fingerprint: Characteristic chemical components, structures, performance differences and specific metabolites
Characteristic pattern: identifiable, unique
Nucleic acid recognition
16srRNA base sequence
rRNA accounts for 80% of total RNA
Many segments are highly conserved
biological evolution timer
phylogenetic map
PCR
Polymerase chain reaction, cell-free cloning, a molecular biology technique that amplifies specific DNA fragments outside cells
Detection of pathogenic bacteria: specific fragments, conservation
Principle: Under the catalysis of DNA polymerase, the parent strand DNA is used as the template, and specific primers are used as the starting point for extension. Through denaturation, annealing, extension and other steps, the process of in vitro copying of daughter strand DNA complementary to the template DNA is carried out.
reaction components
Template DNA: target gene - a gene unique to the microorganism
Primer pair: starting point and end point of replication, specificity of replication
dNTP:A,T,G,C
DNA polymerase
Reaction buffer, magnesium salt solution
advantage
In vitro amplification of specific nucleic acid fragments
Fast, sensitive and specific
Automatic microbial analysis system
Fully automated microbial analysis
Principle: The instrument fixes the biochemical reaction culture medium necessary for bacterial identification on the card. After the culture, the instrument judges the displayed reaction and uses the numerical method to make the determination.
Gram-negative bacteria card, Gram-positive bacteria card, yeast card
API bacterial identification system
immunological methods
Antigen (Ag): A substance that can induce the immune system to produce an immune response and react specifically with the antibodies or effector cells it produces in vivo or in vitro.
Antigenic determinants: active groups on the surface of an antigen
Antigenic properties
Antigenicity: Immunogenicity and Reactogenicity
Heterogeneity: protein, sugar, nucleic acid (chemical composition), 5-10ku poor immune effect (molecular weight)
Antibody (Ab): A type of active substance that can react specifically with the antigen that is synthesized and secreted by B cells when the antigen enters the body. It is mainly found in the serum.
Antigen and antibody detection methods: detection of bacteria or toxins
agglutination reaction
Known Antibodies Identify Antigens
The granular antigen binds to the corresponding antibody, and after a certain period of time in the presence of a dielectric, small agglutinated pieces appear with the naked eye.
precipitation reaction
Neutralization reaction
Labeled Antibody or Antigen Technology
Salmonella 1-2 Experiment
Principle: Performed in two special containers, one containing a selective liquid medium and the other a non-selective semi-solid medium
Antigen and antibody combine to form an immune band visible to the naked eye
Enzyme-linked immunosorbent assay ELISA
Particle antigen: such as bacteria, viruses, etc.
Soluble antigens: enterotoxins, mycotoxins, etc.
qualitative or quantitative
Simple and fast operation
Experimental principle
Enzymes bind to antibodies or antigens
specific reaction between antigen and antibody
Enzyme-catalyzed substrate color development, qualitative or quantitative analysis with naked eyes or instruments
Specificity ➕ Enzyme signal amplification
Required materials
solid support
Enzyme labeled antigen or antibody
Horseradish peroxidase, good stability
alkaline phosphatase
substrate for enzyme action
O-phenylenediamine (yellow)
Tetramethylbenzidine (blue)
type
Direct method, indirect method, sandwich ELISA: determination of particle antigen
Competition method: determination of small molecule antigens such as mycotoxins
sandwich ELISA (sandwich ELISA)
①Coating: Antibody 1 ② Washing: Remove excess and weakly bound reagents ③Add samples to be inspected; ④ Washing; ⑤Add enzyme-labeled antibody 2; ⑥ Washing; ⑦Add substrate; ⑧ Termination reaction
advantage
Can detect multiple samples at the same time, can be automated, simple to operate, and commercialized
colloidal gold chromatography
principle
Limulus (hou) reagent assay
Sensitive method for determination of endotoxins in cell walls of negative bacteria
Principle: Reaction between endotoxin and lysates dissolved in horseshoe crab to form a gel, qualitative or semi-quantitative
Bioanalysis and related technologies
Selection of experimental animals
Sensitivity to test substance, gender, age, weight
Test substance administration method
feeding; diet, drinking water
Oral administration, capsule
Injection: intraperitoneal, subcutaneous
Surgery
Botulinum toxin test
Surgical procedures in animal experiments
ligating ring technology
Sample collection and processing
Sampling principles to follow
Determine the sampling plan: ear level two, level three
Representativeness: conducted randomly, representative of all foods
Aseptic operation to prevent contamination
Maintain the original state: volatilization, temperature
Just-in-time: sampling and sending for inspection
Sampling plan
type
sampling
n: The number of samples that should be collected for the same batch of products
Result judgment
c: The maximum allowable sample value exceeding the m value
m: Limit value of acceptable level of microbial indicators
MThe maximum safety limit value of microbiological indicators
Level 2
Level three
food hazard
Category I hazards, foods for the elderly, infants and young children and foods that may increase hazards before consumption
Category II hazard, food that is eaten immediately, the hazard is basically unchanged before consumption
Category III hazards, foods that have been heated before consumption to reduce hazards
Importance of inspection indicators to food: general, medium, severe