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#7  How to Count Microorganisms

 

 Kenji ISSHIKI
Professor Emeritus at Hokkaido University

Advisor at Japan Food Research Laboratories

Biography of Dr. Isshiki

http://researchmap.jp/isshiki-kenji/

  

1. From Koch’s laboratory

In my column #6, I mentioned that Listeria can proliferate even under such low temperatures as in refrigerators.  How can we count invisibly-small microorganisms like Listeria with the naked eye?


Robert Koch, born in 1843, accomplished many outstanding results and he is called “the founder of modern bacteriology” together with Pasteur.  One of his achievements is a development of culture medium made of solidified agar for the purpose of bacterial research.  His research team had been trying to improve the flaws of liquid medium, and they finally found Eastern agar.

 

Cultivated in an agar-solidified medium, microorganisms begin to proliferate and cluster together, and the size of the group grows to be visible.  This cluster is called “colony”.  The first picture in my column #1 shows a surface of Bacillus subtilis var. natto colony.  It is commonly believed that there are 109 bacterial cells per 1mm3 in a colony.  Picture1 below is from an evaluation process of total viable cells.  This process causes microorganisms in food to form colonies using standard agar medium.  Assuming one bacterial cell forms one colony, counting emerging colonies will reveal the number of microorganisms in food.  This is also called the plate culture viable count method.

 

ishiki71.jpg

 

In food bacteria counting, this plate culture method and MPN method (Most Probable Number method) have been used in many cases.  In the MPN method, diluted samples are inoculated into liquid culture medium divided into 3 to 5 test tubes, and this dilution step is repeated three times.  These methods are complicated to operate and take a number of days and testing staff.  This has been bothering many food manufacturers.  In order to solve this problem, automatic food bacteria count systems have been developed as shown in Table1.  Many food-handling facilities are starting to adopt these systems for their self-imposed hygiene control activities.

 

The plate culture method and MPN method are included in the official bacterial inspection methods stipulated in Food Sanitation Act of Japan.  Also, genetic detection method will find target microorganisms by amplifying their genes.  This method consists of PCR method (gene-amplification method using Polymerase Chain Reaction) and LAMP method (Loop-Mediated Isothermal Amplification at 60-65°C).  These genetic detection methods are designated as notified methods of the Ministry of Health, Labour and Welfare for detecting enterohemorrhagic Escherichia coli.  These genetic methods will detect microorganisms without distinction between life and death.  Therefore, these methods are sometimes used concomitantly with other inspection methods, or bacterial cultivation is conducted before genetic detection process.

 

Table1

Automatic food bacteria count system

Principle

Name of device / kit Target
Oxygen electrode method

DOX

Total viable cells, Escherichia coli, coliforms, lactic acid bacteria, Staphylococcus aureus

Impedance method

RABIT system

Total viable cells, Escherichia coli, coliforms, fungus, etc.

 

Fst GAAD

Total viable cells, Escherichia coli, Staphylococcus aureus, etc.

Bioluminescence method

Lumitester

Total viable cells, aseptic testing of coliforms

ATP method

EnSURE & MicroSnap

Total viable cells, Escherichia coli, coliforms

ATP bioluminescence method

Milliflex Rapid

Total viable cells, fungus

Chemiluminescent method

BactoLumix

Total viable cells, fungus

Flow cytometry method

BactiFlow

D-count

Total viable cells, salmonella, yeast, intestinal bacteria, mold

  Cassette Lab ONE

Total viable cells, yeast

Fluorescence staining method

Bioplorer

Total viable cells, dead cells

  EZ-Fluo

Total viable cells, etc.

  BM-300C 

Total viable cells, dead cells

 

2. From Koch’s laboratory to food manufacturing sites

People in charge of food manufacturing are seriously tackling microorganisms.  Their efforts not to cause trouble to consumers are continued unremittingly, which imposes a huge burden on themselves as a practical problem.  At food manufacturing sites, unlike research laboratories, securing food safety based on 5S program (Sort, Set, Shine, Standardize, and Sustain) is essential.  If their bacterial tests become rapid and easy, many advantages, such as follows, will be realized.

 

Conditions for hygienic food handling can be set based on scientific grounds with less effort and cost.  Conventional methods take so much of them.

 

It becomes quickly obvious if hygienic conditions of food materials and half-finished/finished food products are within the setup acceptable range or not.  Conventional methods take a few days from preparation to obtainment of the test results.

 

When food materials or manufacturing processes are changed, its consequent hygiene can be checked easily.

 

Without a specialist of microorganisms, even part-time workers can conduct the bacterial test after receiving a simple training.

 

 

3. Food bacteria testing system for manufacturing sites

 

An unique bacteria detecting system has been developed and put into practical use from a collaborative research of Daikin Industries, Ltd. and National Food Research Institute of the Ministry of Agriculture, Forestry and Fisheries.  This system adopts a method that determines the amount of oxygen consumed by microorganisms in liquid culture medium using oxygen electrodes (Figure1).  It’s an automatic system  that extrapolates food bacteria count by the amount of consumed oxygen.  Its first-stage development was targeted to determine total viable cell count.  Now it has been improved to evaluate coliforms, Escherichia coli (qualitative), Staphylococcus aureus and salmonella.  This equipment is called “DOX system” from the capital letters of Dissolved OXygen that is evaluated with this system. 

 

 

 Respiration of microorganisms → Oxygen consumption → Change in current value

 

esokuteigenri.jpg

Electric current in proportion to the dissolved oxygen concentration will flow.

 

 

edengraph.jpg

 

  

Figure1

Food bacteria testing using oxygen electrode method (DOX method)

 

 

 

Here are the main features of the DOX system.

(1) Simple operation and user-friendly.

(2) Speedy inspection. The more bacteria exist in the food sample, the quicker it can be detected.

(3) Less manpower and time.

(4) Low running cost.

(5) Concurrent testing of multiple samples.

(6) Test results are stored in the database automatically.

(7) Space-saving.

 

For more information, visit the website of Bio-Theta, Ltd. ( http://www.bio-theta.co.jp/ ), a manufacturer specialized in the DOX system. 

 

Now, how should we evaluate the “Vitamin I (love)” which is essential in food handling as I mentioned in my column #3?

 

 

References (all in Japanese)

Yoshikura, H. Records of Lectures on Microbiology. Japanese Society for Virology. http://jsv.umin.jp/microbiology/

Amano, Y., et al.  "Sansodenkyoku wo Mochiita Ippanseikinsuu Kensa Kanijinsoku-ka (Simplifying & Accelerating the Evaluation of Total Number of Microorganisms Using Oxygen Electrode)".  Journal of the Japanese Society for Food Science and Technology 48 (2001): 94-98.

S. Kawasaki, et.al.: Comparison of traditional culture method with DOX system for detecting Coliform and Escherichia coli from vegetables, Food Sci.Technol.Res., 9, 304-308(2003)

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