Colony Counting: Standard Operating Procedure for Colony Counter

A colony counter is an instrument used to count colonies of bacteria or other microorganisms growing on an agar plate.

The counting may be performed:

1. manually – were merely lighted surfaces on which the plate is  placed, with the colonies marked off with a felt-tipped pen on the outer surface of the plate while the operator kept the count manually

2. automatically – to count the colonies electronically, by identifying individual areas of dark and light accor-ding to automatic or user-set thresholds, and counting the resulting contrasting spots                                                          

Such counters are used to estimate the number of microorganisms within a liquid or product. An appropriate dilution, or several dilutions within the estimated appropriate range, is poured or  spread using sterile technique on the agar plate, which is then incubated under the appropriate conditions for growth until individual colonies appear. Each colony marks the spot where a single organism was originally placed, thus the number of colonies on the plate equals the number of organisms within the volume of liquid spread on the plate. That concentration is then extrapolated by the known dilution from the original culture, to estimate the concentration of organisms within that original culture. The maximum number of colonies which may be effectively counted on a single plate is somewhere between 100 and 300, depending on the size of the colony and the type of organism. The colony counter should be able to count bacteria on “spiral”,  spread”, “settle” and “pour plates” methods.

•  Standard Operating Procedure

- TITLE

Colony counting by manual colony counter

- SCOPE

To standardize the reading and counting of Colony Forming Unit (CFU) on agar plates

- GLOSSARY

Aerobic Plate Count, Agar, Agar plate, Bacteria , Colony Forming Unit, CFU, Colony, Dilution factor, Incubation, Medium, Petri dish, Pin-point, Poured method, Spread method, Spiral method, Sterilization

- RESPONSIBILITY

Microbiological Laboratory Manager

- SAFETY

Individual Protective Devices for biohazard safety

- STANDARD

ISO Standard

- MATERIAL

- Colony Counter 

- Electronic Marking pen

- Petri dishes

- Autoclavable Plastic bag

Petri plate

Pen electronic colony counter

Viewer

- PROTOCOL

1. Turn on the electric power to light the LED and fluorescent lamp.

2. Place the updown Petri dish on the surface of the counter by adjusting the dish holder.

3. Adjust the focusing arm with the lens to the desired angle, if small colonies are to be counted.

4. Begin counting by touching the Petri dish with the marking pen. Every time a colony is marked, the electronic colony counter pen will register the count.

5. If the colonies are numerous and evenly distributed on the agar surface, it is possible to reduce the time and to count just to opposite segments of the plate and then calculate the total number of the cfu (multiplying for the number of segments).

6. When the counting of the Petri dish is completed, take note of the counted number in the report paper and replace the Petri dish with the next one.

7. At the end of activity, turn off the unit.

8. Guidelines for calculating and reporting.

APCs: Official Methods of Analysis  does not provide guidelines for counting and reporting plate counts, whereas Standard Methods for the Examination of Dairy Products, 16th ed. presents detailed guidelines; for uniformity, therefore, use APHA guidelines as modified (6,8). Report all aerobic plate counts  computed from duplicate plates. For milk samples, report all aerobic plate counts computed from duplicate plates containing less than 25 colonies as less than 25 estimated count. Report all aerobic plate counts  computed from duplicate plates containing more than 250 colonies as estimated counts. Counts outside the normal 25/250 range may give erroneous indications of the actual bacterial composition of the sample. Dilution factors may exaggerate low counts (less than 25), and crowded plates (greater than 250) may be difficult to count or may inhibit the growth of some bacteria, resulting in a low count. Report counts less than 25 or more than 250 colonies as estimated aerobic plate counts (EAPC).

Use the following guide:

1. Normal plates (25/250). Select spreader-free plate(s). Count all colony forming units (CFU), including those of pinpoint size, on selected plate(s). Record dilution(s) used and total number of colonies counted.

2. Plates with more than 250 colonies. When number of CFU per plate exceeds 250, for all dilutions, record the counts as too numerous to count (TNTC) for all but the plate closest to 250, and count CFU in those portions of plate that are representative of colony distribution. See ref. 2 for detailed guidelines. Mark calculated APC with EAPC to denote that it was estimated from counts outside 25/250 per plate range (see D-3).

3. Spreaders. Spreading colonies are usually of 3 distinct types: 1) a chain of colonies, not too distinctly separated, that appears to be caused by disintegration of a bacterial clump; 2) one that develops in film of water between agar and bottom of dish; and 3) one that forms in film of water at edge or on surface of agar. If plates prepared from sample have excessive spreader growth so that (a) area covered by spreaders, including total area of repressed growth, exceeds 50% of plate area, or (b) area of repressed growth exceeds 25% of plate area, report plates as spreaders. When it is necessary to count plates containing spreaders not eliminated by (a) or (b) above, count each of the 3 distinct spreader types as one source. For the first type, if only one chain exists, count it as a single colony. If one or more chains appear to originate from separate sources, count each source as one colony. Do not count each individual growth in such chains as a separate colony. Types 2 and 3 usually result in distinct colonies and are counted as such. Combine the spreader count and the colony count to compute the APC.

4. Plates with no CFU. When plates from all dilutions have no colonies, report APC as less than 1 times the corresponding lowest dilution used. Mark calculated APC with asterisk to denote that it was estimated from counts outside the 25/250 per plate range.

- NO CONFORMITY

When plate(s) from a sample are known to be contaminated or otherwise unsatisfactory, record the result(s) as laboratory accident (LA).

• Reference

- Health Protection Agency UK – Standard Methods for Aerobic Colony Count   

- U.S. FDA. Bacteriological Analytical Manual. Aerobic Plate Count.

Ten steps for a perfect “SOP”

The production of a correct “SOP” document is an effective “piece of paper” for managers, laboratory technicians, servicing people to apply the correct GLP, GMP, to improve the performances, to avoid mistakes, to motivate workforces.

Ten steps for an ideal “SOP”

• 1. Goals to be reached

SOP work best when they are designed to achieve specific results. Decide what business goals will be achieved through better management with SOPs and how those goals will be measured.

• 2. Responsibility

A specific person or team must be dedicated to each single SOP.

• 3.The first draft

Make a detailed list of the steps in the order that they should be done.

A simple way to get started is to observe someone performing the process as it now exists and write down everything that that person does. This list is now a draft of the procedures. If the procedure needs to appear as a flowchart, start with the most reasonable beginning point. Draw the decision that a worker will need to make and actions that follow each decision. Do not try to be perfect with the first draft, because it is very likely that you will need to make many revisions.

• 4. Paragraphs of the SOP

An example of the SOP composition could be:

Title. Scope. Glossary. Standard / References. Responsibility. Safety. Material. Protocol. Non Conformity. Corrective Actions.

• 5. Internal Review 

Provide each worker who performs the procedure with a copy of the draft SOP. Ask the workers to review and suggest changes that will make the procedure easier to understand or more accurate or will improve performance. Assure the workers that their input is important and will be used.

People are much more likely to accept and use a SOP if they feel a sense of ownership in it. Another reason to involve the workers is that they are likely to have good ideas.

• 6. External review

Provide advisers with a copy of the SOP draft. Ask them to suggest any changes that will make it clearer and more effective. Revise the procedure as necessary to incorporate their input.

• 7. Test

There is only one way to be absolutely certain that a procedure is well written and performs as expected. Have someone test the procedure by performing each steps exactly as it is described while the procedures writer watches. Have a person not familiar with the work follow the procedure. Any steps that cause confusion or hesitation for the test worker should be re-considered.

• 8. The final draft in appropriate location

Produce a final draft and post it in an appropriate location. The workplace is one essential location. A master SOP file should be kept in a central location so workers can review little-used SOPs when necessary. It is essential to keep SOPs up to date.

• 9. Training

Train everyone as necessary to follow the procedure exactly. Otherwise, individuals will interpret the meaning of procedures in different ways, leading to inconsistency in work routines and performance.

The trainer will explain and demonstrate both why and how each step in the SOP is performed and then give the learner a chance to practice.

• 10. Audit

An audit will show whether the procedure is being adhered to and whether the objective are being met.

The SOP should be audited when dispensing errors or “near misses” occur, to identify ways of preventing their recurrence.

The correct SOP

SOPs are typically created using Microsoft Word, printed out, and stored in three-ring binders.

The binders are then routed physically from one approver to the next. 

Once approved, the content of the binder is photocopied so every affected department will have one binder handy.

The possible negative aspects of a SOP

The most common negative aspects are: 

(a) Inefficiency

(b) Poor Communication

(c) Lack of Training

(d) Ineffective Documentation

(e) Poor Revision Control

(f) Poor Collaboration

(g) Lack of Visibility

(a) Inefficiency

The process of routing, reviewing, and approving a SOP can be slow and inefficient.  This is especially true if an organization generates hundreds of SOPs and the people who need to review and approve them are in different location. It is likely the SOPs may get lost while being routed, either buried in someone’s messy desk or computer “in” box. Search of SOPs would entail sorting through voluminous paper work. Updating SOPs would be equally slow.

(b) Poor Communication

In a manual system, quality processes are not connected, making it hard for people who operate those processes to communicate. To speed up the approval of a SOP, the originator needs to make phone calls, send e-mail messages or remind approvers in person, and that is just one SOP! Multiply the number of phone, e-mail, and personal reminders a dozen times for an employee responsible for a dozen SOPs.

SOPs are likely to be updated, but people affected by the changes may not be notified in a timely manner.

(c) Lack of Training

Employees may be using new SOPs, but they may be doing it poorly without the appropriate training.

(d) Ineffective Documentation

Effective implementation of SOPs has a direct impact on product quality, and that is why it is critical for organizations to capture and up-to-date information in SOPs.

It is sometimes difficult to rely on employees to always remember to document any changes in an SOP, including who made the change, why, and when.

(e) Poor Revision Control 

After SOPs have been revised and approved, old documents may re-surface on the shop floor. Another problem is that employees may use documents that have not been approved simply because they are able to grab that binder sitting on someone’s desk. In both cases, using uncontrolled SOPs could lead to nonconformity.

(f) Poor collaboration

Creating and updating SOPs usually require collaboration. Collaboration is relatively easy when employees are based in the same facility and with the same schedules. Collaboration is quite difficult among employees with varied schedules and responsibility and who are based in different facilities.

(g) Lack of Visibility

Paper-based processes are not connected, making it difficult for management to monitor the effectiveness of  SOPs and make timely decisions.

Conclusions

For FDA-regulated and ISO-certified companies, SOPs are more than just a tool of operation. SOPs directly affect product quality, and therefore, compliance. Development of effective SOPs and implementing them properly require commitment from the entire organization. Choosing the right software solution that will facilitate and improve SOP management should be a part of this commitment. Once established, an efficient and effective SOP management system will serve as a foundation for an organization’s sustainable compliance and long-term success in the market.

Reference

SOP Management as a Compliance Tool in FDA and ISO Environment – 2006 MasterControl Inc. 

White Paper. Page 4 and 5. www.mastercontrol.com