| Why count microorganisms in foods? | - gives product history
- predict shelf life: inverse relationship between initial microbe population and shelf life
- predict safety |
| Why look for specific organsims? | - indicators of specific problems
- some pathogens known to contaminate product
- presence of beneficial microbes |
| Important characteristics of assays | - accurate
- sensitive (esp. for pathogens)
- reproducible
- cost effective
- rapid (save time and money) |
| How to make plate counts more specific | - selective agar
- differential agar
- selective differential agar
- specific incubation atmosphere: aerobic, anaerobic, microaerophilic
- specific incubation temperature: psychrotrophs (20C), mesophiles (35-37C), thermophiles (50C) |
| Selective agar | Media utilizes selective agents where target organism is resistant to that agent. Ex: chemicals, salt, antibiotics, dyes |
| Differential agar | Media utilizes unique metabolic capabilities of an organism so they show up differently in the agar. The target organisms will metabolize or change the medium to distinguish themselves i.e.. color change. |
| Four basic methods to determine "total" number of microorgansims | - Standard plate count (SPC)
- Most probable number (MPN)
- Dye reduction technique
- Direct microscopic counts (DMC) |
| Conventional SPC | - most widely used method for determining CFU in food product
1) blend sample
2) serial dilution - usually 10 fold, dilute out inhibitors (natural, preservatives)
3) plating
4) incubation
5) count - multiply CFU by reciprocal of dilution factor |
| Factors impacting SPC results | - single vs. composite sampling
- distribution of microorganisms in food
- types of microorgansims in food (oxygen tolerance)
- nature of food material (fatty - add emulsifier, acidic- add buffer)
- nutritional adequacy of plating medium
- pH, aw, redox potential of plating medium
- incubation temp. and time
- type of diluent used
- competing or antagonistic microorgansims |
| Types of SPC diluent | - phosphate buffer saline (PBS)
- saline (0.85% NaCl)
- 0.1% peptone
- BPW |
| Spiral Plate Method | - aka automated standard plate count
- known volume of sample dispensed on rotating agar plate in spiral pattern
- volume of sample decreases as spiral moves toward edge of plate
- laser beam counter relates area of plate to sample volume to calculate colonies |
| Spiral Plate Method - advantages to SPC | - less agar media: 2-3 dilutions on 1 plate
- fewer petrie dishes, dilution blanks, and pipettes required
- 3-4 x more samples/hr tested
- 50-60 more plates/hr prepared
- little training required |
| Spiral Plate Method - disadvantages to SPC | - food particles can block dispenser
- more suited for liquid foods
- expensive equipment |
| Membrane Filters | - food sample pre-filtered to remove large particles (10 um pore)
- sample goes through 0.22/.45/.6um filter that allows diluent to pass and retains bacteria
- filter is stained and examined microscopically or placed on agar for colony formation
- techniques: direct epifluorescent filler technique (DEFT), microcolony DEFT, and hydrophobic grid membrane filter (HGMF) technique |
| Direct Epifluorescent Filter Technique (DEFT) | - fluorescent dye: acridine orange, stains DNA and mRNA
- fluorescent microscope
- food homogenate pre-filtered through 10um pore size nylon filter, then through nucleopore polycarbonate filter (0.22/0.45 um pore size)
- filter stained, dried, and counted
- can detect live and dead cells with LIVE/DEAD BacLight Viability Assay |
| Bacterial cell count in DEFT/using microscopy | Need: area of microscopic field, surface area of filter, avg. bacterial cell count per MF, volume/weight of food filtered
Calc: (#MFs per filter * avg cell count/MF) / volume or weight |
| Microcolony Direct Epifluorescent Filter Technique (Microcolony DEFT) | - filter with trapped cells placed on agar medium
- incubation for 3-6h
- filter stained with fluorescent dye
- microcolonies viewed with EF microscope |
| Hydrophobic Grid Membrane Filter | - up to 1g food homogenate filtered through membrane filter (0.45 um pore) with 1600 wax grids
- filter placed on agar medium surface
- incubation (24-48h); counting of positive grids
- calculate most probable number
MPN = 1600 * 2.303 *log10(1600/(1600-c))
c = number of positive grid cells |
| Hydrophobic Grid Membrane Filter advantages | - filtration removes undesirable substances
- better detection limits than SPC
- stressed cells can be resuscitated on non-selective agar then transferred to selective agar
- colonies stick to HGFM, so other procedures can be applied |
| Petrifilm | - dried culture medium between two films
- tetrazolium dye in culture medium
- 1ml sample deposited and spread over medium by pressing
- incubation
- count colonies (show up red) |
| Roll tubes | - used for anaerobes
- known volume of melted agar added to tube
- food sample added
- tube flushed with N2 gas or N2 and CO2, then closed
- rolled in ice slush, incubated for 24-48h
- colonies in agar on tube wall counted |
| Agar components in roll tubes | Resazurin (dye)
- REDOX indicator
- pink (oxidized); colorless (reduced)
- added to agar before autoclaving
Cysteine
- reducing agent
- decreases REDOX potential of agar
- added to agar after autoclaving |
| Most Probable Number (MPN) | - statistical-based method to give MPN of organisms present in a sample
- prepare serial dilutions of food sample
- inoculate 9 (3 tube method) or 15 tubes (5 tube method)
- incubate tubes
- count positive tubes and use table to find MPN |
| Most Probable Number (MPN): advantages and disadvantages | Advantages: simple, flexible (use various media)
Disadvantages: lots of glassware req., lack of precision, no isolated colonies |
