Quantitative Microbiology

Now I'm going to look at the detection of water borne and food borne bacteria. Methods of detection either involve:

1. Detecting specific pathogens.
2. Establishing the general level of contamination of food or water. 

Detecting Specific Pathogens 

Required when we suspect infection with a specific pathogen. 

For rapid detection the following method may be used:

• Pre-enrichment for the specific pathogen before culture or before processing for PCR using specific primers. 
• Selective enrichment culture for the specific pathogen. This may be followed by PCR using specific primers. 
• Selective differential culture for the specific pathogen. 
• Presumptive identification.
• Biochemical identification.
• Serotyping/phage typing. 

Bacterial product detection:

• Pre-formed toxins- such as staphylococcal enterotoxin, and clostridial toxins.
• Specific antigens. 

Bacteria Associated With Food/Water Borne Infection

Salmonella on Brilliant Green Agar

• Salmonella enterica (selenite broth & Brilliant Green media). 
• Campylobacter jejuni/coli (skirrows broth & selective media microscopy for jejuni and genotyping)
• E.Coli (TSB broth & SMAC selective indicator media and genotyping). 
• Staph Aureus- detect toxins, baird-parker selective indicator media. 
• Clostridium Botulinum- detect toxins A, B, E and F. Antigen ELISA assays or bioassays e.g. cytotoxicity or detect genes encoding toxins. 
• Listeria Monocytogenes (listeria enrichment broth and culture in selective media and genotyping). 

Generally each bacteria is treated with a pre-enrichment method e.g. transport medium magnetic beads. The bacteria then either undergo enrichment with a broth, presumptive identification via culture in indicator media or identification (biochemical tests, serology, and phage/serotyping in the case of salmonella). This applies to the first 3 bacteria and last bacteria. 

Establishing the General Level of Contamination in Food/Water

1. Using quantitative bacteriology. 
2. Detecting indicator bacteria e.g. E.coli.

Quantitative Bacteriology

Provides good information about the levels of contamination. Perceived that more testing for food pathogens will lead to greater food safety. It can lead to better guidelines for food safety and intervention and greater public confidence in food processing. 

Generic principles:

• In unicellular organisms growth is measured in cell mass and number. 
• Mass of cells increases continuously whereas increase in cell number is discontinuous and occurs as a result of cell division. 

Methods:

Direct cell counts- all cells counted in a large square and the number averaged across several squares.                

• Light microscopy uses a counting chamber with known liquid volume but it is not very practical for small bacteria. It does not differentiate live from dead cells and requires a high concentration of cells.
• A counter counter is widely used for counting animal cells e.g. blood cells and not practical for small cells.
•  Direct cell counts is done by flow cytometry. The basic principle is flow of narrow stream is scanned by laser light at various frequencies and angles. It permits estimation biomass by light scattering or staining of chemical components like DNA with fluorescent dyes. It is used for identifying different cell subsets and is widely used in animal cells. It can separate cells but is not very practical for quantitative biology. 

Total viable colony counts establishes the number of colony forming units (CFUs) per volume or weight of the original sample according to standard methods. 

• Ten fold serial dilutions of a sample.
• Inoculate agar plates in triplicates for each dilution.
• Incubate at 37 degrees for 24-48 hours. 
• Count the number of colonies on each plate starting with the highest dilution. 

To minimise error 2 or 3 must be used for each dilution. 30-300 colony plates yield the most reliable results. The same method can be used for detecting indicator bacteria. The problems associated with this method is that many serial dilutions are required, they require special statistics, lower counts are difficult to interpret, and it is time consuming sometimes taking days before an answer. 

Most probable numbers is widely used to establish the level of contamination in water. 

• Probably number of organisms per 100ml of water.
• Inoculate three sets of tubes with a ten fold difference in inoculum. Incubate @ 37 degrees for 24-48 hours. Score each set for the number of positive tubes. 
• Calculate the most probably number using standard tables.

Light scattering (optical density) is a method which measures the amount of light scattered by a suspension of cells in a liquid. 

• Spectrophotometer to measure turbidity which is affected by the number of bacteria present. 
• The output is optical density (OD). 

Measuring cell components (metabolic activity). 

Measuring genetic material (real time quantitative PCR) is detection of bacterial DNA. 

• Very sensitive- 1 copy- 10 copies of DNA.
• Detects organisms that cannot be isolated in culture.
• Can be very rapid <24 hours. 
• It is technically difficult.
• It is expensive.
• There is a contamination risk.
• It requires rigid quality control.