Colony Counter – Principle, Types, Parts, Operating Procedure

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To make colony counts of Proteus in food and subsequently to identify them, the food is emulsified and then diluted in a suitable sterile isotonic diluent and then is plated out on culture media with a dry surface that do not permit swarming growth.

What is Colony Counter?

• In clinical, food, dairy, and pharmaceutical microbiology, taking a total microbial count or total viable count is a standard practise.

• Traditional manual methods for counting bacterial colonies (or colony forming units) are laborious, time-consuming, and require a high level of focus.

• Colony counter offers an alternate, sustainable solution to this issue by facilitating the rapid and precise counting of colonies.

• A colony counter is an equipment used to count colonies of microorganisms growing on agar plates.

• There are numerous types of colony counters available for counting bacteria and yeast colonies rapidly and precisely. Some of these colony counters are manually operated, while others are automatically operated.

Type of Colony Counter

There are two sorts of colony counters, including;

Manual colony counters: The manual colony counter procedure is straightforward: Petri plates are placed inside the colony counter and illuminated and enlarged. A digital display will show the total number of discovered colonies while operators can mark them with a specially designed marker.

Automatic colony counters: A colony counter that is automated reduces counting time from minutes to seconds, eliminates recording mistakes, and standardises counts between users.

Manual colony counters

• Manual and semi-automatic counters rely on a technician’s ability to see colonies clearly and label them with a special sort of pen on the plate’s outer surface.

• The machine maintains track of the number of marked colonies. Although this method is somewhat effective, it is still time-consuming, laborious, and error-prone.

Parts of manual colony counters

• Light source: A source of backlight is utilised for illuminating reasons.

• Auto marker probe pen: When a counter pen is pressed against a colony, a beep is heard and the count is shown on a computer screen.

• Digital display: It shows how many times the counter pen has been used.

• Lens: It helps make things bigger.

Manual colony counter Principle

• A model of a manual colony counter works by putting a Petri plate on an electronic pressure pad with lights and marking each colony by touching the plate with a felt tip pen.

• The digital display shows a number based on how hard you touch it. The pressure can be changed depending on what is needed.

• With this model, you won’t miss counting colonies or count them twice. The instrument also comes with a Wolfhuegal graticule, a segmentation disc, and centering adapters for 50-90 mm plates.

• This model has some extra features, such as a dark background for colonies that are translucent, glare-free lighting, and a built-in average function that lets you count colonies on more than one plate.

• The software makes the average number of colonies and changes the background with a toggle switch. Using USB connectivity, the data about the counted colonies is sent to the computer.

• Colonies can be small and crowded, which makes it hard to count.

• So, breaking the counting up into small squares and using a magnifying glass on a flexible arm to see the colonies better makes the job a little bit easier.

• This method for counting colonies has a very low output and takes a lot of time. Also, the number of colonies can be different when more than one technician counts.

Operating Procedure for Manual Colony Counter

• Press the On/Off Switch to turn the instrument ON.

• Put the Petri Plate on top of the grid made of glass.

• Remove the cap from the Pen and press hard, keeping the Pen straight, on the Petri Dish where a Bacteriological Colony is located. The Counter will record a number, make a beep, and mark the Petri dish with a dot of ink. Keep going until all of the colonies have been counted this way.

• As each colony is marked with ink as it is counted, it is impossible to miss a count or count it twice. When there are no more numbers to count. Take note of what the counter says.

• The COUNT push button switch, which is next to the RESET switch, can be used to count plates with few people on them or for other counting tasks.

• Switch to “decrement” to drop the count by one.

Limitations of Manual colony counter

• Slow going

• High risk of mistakes by people

• slows things down

• depends on sight

• No way to find plates or see pictures of them.

• needs to be put into LIMS by hand

• LIMS must be used to make reports.

• Lack of being able to track

• There are differences between technicians

Automatic Colony Counters

The automatic bacterial colony counter makes it easy to count colonies by using image processing algorithms like grey scaling, thresholding, filtering, etc.

Automatic Colony Counters Principle

• A fully automatic colony counter uses a document scanner, digital camera, webcam, charge-coupled device (CCD), or video equipment to take pictures of the colonies.

• Taking a picture of the colonies on a Petri dish is the first step in counting them. The image is then turned into a digital file using software.

• The last step is to use a single/multi-threshold segmentation procedure to separate and find the colonies in the digitised image.

• The method for counting colonies will save time if it can be done automatically.

• The labs that don’t have a colony counter can send the images to the lab that has the software for analysing them over the internet.

• In an automated system, the contrast between the objects and the background and the transparency of the background vary a lot.

• To make counting colonies easier and more accurate, one of the following ways of lighting is chosen; Transmission method: For everyday objects with high contrast and backgrounds that are mostly clear. Dark-field method: For things that stand out against a dark background. Reflection: For objects with low contrast and backgrounds that are mostly clear.

• With its 1.4-megapixel CCD, the modern tool can take HD (high definition) colour pictures.

• In the same way, a typical automated colony counter is made up of long-lasting red, green, and blue LEDs that help get good colour images with bright contrast and no chromatic aberrations.

• For effective bright field and dark field exposure of the sample platform, you can use a black, white, or clear background.

• Both 55-150 mm round plates and 150 mm x 150 mm square plates can be used. It can measure zones as small as 0.1 mm or colonies as small as 43 m.

• Because they are clear and reflect light, it is hard to light up Petri dishes without shadows or reflections.

• Multiple lights could make artefacts on the edge of the agar, which are likely to be counted as a colony. It makes counting wrong, which affects how precise the instrument is. Interscience for Microbiology has made a white dome that diffuses light in all directions so that there are no shadows or reflections. This feature is built into their Scan® 4000 model.

Parts of Automated Colony Counter

1. Culture dish

• The automatic colony counter can be used with many different types of culture dishes, like traditional plate inoculation, spiral inoculation, etc.

• Second, the standard sizes of its culture dishes are 90mm and 55mm in diameter.

2. Light source

• The automatic colony counter uses a long-lasting LED light source as its light source.

• There are four different ways to combine a light source with a background colour.

• Optional smart remote control multi-color light source background, which makes it easier to keep track of the different types and colours of culture media.

3. Imaging

• For imaging, the traditional artificial colony counter uses a magnifying glass with 3–6 times magnification.

• The automatic colony counter has a camera function, and the CCD specifications are getting higher and higher.

• The HCC-90A model that is now being sold has 5 million pixels, a colony resolution of 0.1mm, and a much clearer image.

4. Image processing

• The automatic colony counter is very powerful in its ability to process images.

• Traditional colony counters don’t have functions like background processing, colour marking, interference correction, colony enlargement, area calculation, and so on.

5. Database

• In terms of database processing, automatic colony counters are better than traditional colony counters because they increase data storage, intelligent query, data export, etc. Some can also set operator permissions, data modification permissions, etc., to make the measured data safer. You can also make changes online and print reports at the same time.

Operating Procedure

• Counting the colonies electronically by distinguishing individual areas of dark and light based on automatic or user-set criteria and counting the resulting areas of contrast.

• These counters are utilised to estimate the quantity of microorganisms contained within a liquid or product.

• Using sterile procedure, an acceptable dilution or many dilutions within the anticipated appropriate range are poured or spread on the agar plate, which is then incubated under the appropriate growth conditions until individual colonies emerge.

• Each colony identifies the original location of a single organism, therefore the number of colonies on the plate corresponds to the number of organisms in the volume of liquid scattered across the plate.

• This concentration is then extrapolated by the original culture’s known dilution to approximate the concentration of organisms in the original culture.

• Depending on the size of the colony and the type of organism, the maximum number of colonies that may be effectively counted on a single plate is between 100 and 300.

• The colony counter must be capable of counting bacteria using the “spiral,” “spread,” “settle,” and “pour plates” techniques.

Automated Colony Counter Advantages

• Saves time

• Increases the total number of samples each hour

• Improves accuracy

• Standardises results

• Eliminates human error

• Heightens sensitivity to the presence of smaller colonies

• Automatic data transfer to database / LIMS

• Images and data on counts are stored for use in traceability.

• Complete audit trail and report production (both PDF and Excel formats available).

• Can link to barcode readers to avoid data input errors

Precautions

To avoid contaminating the petri dish with pen ink, counting can be performed using one of the following methods:

• If the Petri Plate is carefully plated (thin layer), it can be inverted and marked on the bottom (which is now on top)

• The glass cover can be placed over the plate and be used for marking. In this instance, however, because there is a space between the media and the marking, the counting must be performed in a single sitting without adjusting the viewing angle or moving/tilting the plate or its cover. Keep the probe’s cap on while not in use.

• Standard plates (25/250) Select a plate with no spreader (s). Count all colony forming units (CFU) on the specified plate, including those of pinpoint size (s). Note the used dilution(s) and the total number of colonies enumerated.

• Plates containing around 250 colonies. When the number of CFU per plate surpasses 250, for all dilutions, record the counts as too numerous to count (TNTC) for all save the plate closest to 250, then count CFU in representative areas of the plate. Mark APC with EAPC to indicate that it was estimated from counts outside the range of 25/250 per plate.

• Spreaders. There are typically three distinct types of spreading colonies: 1) a chain of colonies that are not too clearly separated and appear to be the result of the disintegration of a bacterial clump; 2) one that develops in a film of water between the agar and the bottom of the dish; and 3) one that develops in a film of water at the edge or on the surface of the agar. Report plates as spreaders if (a) the area covered by spreaders, including the total area of repressed growth, exceeds 50% of the plate area, or (b) the area of repressed growth exceeds 25% of the plate area. When counting plates containing spreaders not deleted by (a) or (b), count each of the three separate spreader types as one source. If only one link exists for the first type, it should be counted as a single colony. If many chains appear to emanate from independent sources, each source should be counted as a single colony. Each individual growth in such chains should not be counted as a separate colony. Types 2 and 3 typically produce separate colonies, which are counted as such. Combining the spreader and colony counts to determine the APC.

• Plates lacking CFU. When plates from all dilutions lack colonies, the APC should be reported as less than one times the lowest dilution used. Indicate with an asterisk if the APC was estimated from counts outside the 25/250 per plate range.

• The unique characteristic of this instrument is that it is compatible with any marker pen with a hard point. You are not restricted to utilising a specialised pen.

Why switch to an automated colony counter?

In an effort to standardise count results and enhance traceability, a growing number of laboratories are adopting automated and semi-automatic colony counters. These methods not only accelerate the testing process, but also generate a consistent pattern of data that removes human error and variation. In recent years, there has also been a push for ‘lean laboratory’ efforts, which aim to enhance throughput and efficiency by focusing on value-added activities and minimising inefficient lab operations. Colony counting is one of the easiest processes to automate, as automated colony counters can analyse up to 75 plates in 5 minutes. This can free up a substantial amount of time for laboratory analysts to devote to “value-added” duties and initiatives.

What is the Importance of Colony Counting?

Microorganisms are extremely difficult to count directly, which is one of the primary reasons colony counting is so important. This is nearly entirely attributable to their size; they are simply too little to calculate with adequate precision. Consequently, calculating CFUs is the best alternative. Once an exact plate count is obtained, not only do you have a better sense of how many microorganisms are present, but you also know how many COULD be there in the future.

Counting colonies is a practise utilised in various businesses, including food and beverage companies. The FDA strictly regulates the presence of bacteria and other germs; therefore, understanding your colony counts ensures that you’re releasing harmless items. This also ensures that your organisation is up-to-date on any compliance laws or regulations it must observe.

In a more medical atmosphere, there are also a number of significant applications. Knowing more about the concentration of microorganisms in the blood might better tell medical practitioners about a patient’s condition. If you know the concentration of germs in a person’s blood, you may also monitor the progression of an infectious disease and their immune system’s strength.

References

Gupta, Surbhi & Kamboj, Priyanka & Kaushik, Sumit. (2012). Methodology for Automatic Bacterial Colony Counter. 10.1007/978-3-642-30157-5_56.

Colony Counter, Automated. (2019). Compendium of Biomedical Instrumentation, 491–494. doi:10.1002/9781119288190.ch95

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