Stripe Plate Method: Principle, Types, Methods, Applications (2023)

I bringmeans literally "a long thin line”: and the scratch plate method is a microbiological culture technique in which a sample is spread out in a petri dish in the form of a long, thin line on the surface of a solid medium.

What is the scratchboard method?

The scratch plate method is a microbiological laboratory technique for isolating pure cultures and/or obtaining well isolated bacterial colonies from a mixed population. It is mainly used to obtain pure cultures of bacteria; However, yeasts can also be isolated by this method. It is one of the most commonly used aseptic techniques in microbiology for isolation and propagationbacteria🇧🇷 It is a mechanical isolation technique used in microbiology, commonly known as "Methode Null“.

This procedure dilutes the bacterial load on the surface of the agar medium successively as streaking progresses and finally only a few bacterial cells are inoculated at the end, giving well isolated colonies on the last streaks. Therefore, this method mechanically isolates the bacteria from a mixed population of the same or different species. After inoculation, the same types of colonies are seen in the terminal streaks when the sample contains only one species, while different types of colonies are seen when the sample contains different species.

This is a very old method, used in microbiology since the time of Robert Koch. This method was developed by Loeffler and Gaffky in Koch's laboratory and was first used to serially dilute bacteria on the agar surface to obtain well-isolated colonies. Since then it has been used as a very important tool in bacteriology.

It is a very simple and reliable aseptic technique that uses tools such as cotton swabs, wooden or plastic sticks and toothpicks or loops to dilute and spread the sample on the surface of certain pre-sterilized solid culture media. The sample used can be a suspension or colonies from the agar surface. Well isolated colonies can be obtained from a successful striatum, allowing to describe the colony character of the organism in this specific culture medium and under these conditions.

Goals of the strip plate method

1. To obtain a pure culture of bacteria from a mixed culture
2. To obtain well isolated colonies
3. spreading bacteria

beginningScratch-Board-Methode

The streaking method is based on dilution during the process of mechanically spreading the inoculum on the surface of the solidified culture medium to obtain well isolated colonies from the sample in the final streaks. The sample can be a colony on a solid medium or a suspension in broth. Samples are taken with different tools, mainly with a sterile inoculation loop or a swab. The sample is placed on a sterile surface of solid medium at one edge of the petri dish and a swab is taken. With the tool, the smear is sequentially distributed in different patterns on the agar medium. As striation progresses, the inoculum is gradually diluted to the point where the bacterial cells separate as individual cells or as a colony forming unit (CFU) within a few millimeters. When these inoculated plates are incubated, isolated bacteria or a CFU will result in a well isolated colony. This allows us to obtain a pure culture and describe the morphology of the colony of the organism.

Types of strip plate method

Based on the stripe pattern, the stripe plate method can be classified into 4 types, namely quadrant stripe, T-stripe, continuous stripe and radiating stripe.

1. Quadrant stripe

It is the most common and preferred method, seeding four equal sections of the agar plate. It is also known as the "four quadrant sequence" or "four sector" or "four way sequence" method.

In this process, each board is divided into four equal sectors and each adjacent sector is scratched sequentially. The sector that is scratched first is called the first sector or first quadrant and has the highest concentration of inoculum. Gradually, the inoculum is diluted in the second, third, and fourth quadrants. When the fourth quadrant has hatched, the inoculum is very dilute, resulting in isolated colonies after incubation.

Most often, a discontinuous form of scratching follows, in which the loop at the end of each quadrant is sterilized before the next quadrant is scratched. However, if the bacterial load is very low (or very dilute), the continuous mode can also be used. With the latter, it is not necessary to sterilize the loop at the end of each quadrant.

Although this is the most popular method, it limits us to using a single sample per plate. If two or more samples are tested on a single 10 cm plate, this method is not suitable.

2. T-strip

It is another scraping method in which the agar petri dish is divided into three sections and each section is scraped. Therefore, this method is also known as the "three-sector sequence" method.

The media is divided into three sections by drawing a letter "T" and hatching each adjacent section in turn. At the time of sowing the last section, the inoculum is diluted enough to produce isolated colonies after incubation. A batch form of incubation is mainly followed; However, a continuous form for the highly diluted sample can also be used.

Similar to quadrant hatching, this method makes it difficult to grow two or more specimens on a single 10 cm plate.

3. Continuous scratches

It is another commonly used method, in which an inoculum is evenly distributed in a single continuous motion from the starting point to the middle of the plate. There is no need to split the plate and sterilize the handle during the process. It's easy and quick; However, the problem is that we can only use it when the inoculum is very dilute, or we simply have to propagate a pure culture instead of isolating it.

We can divide the 10 cm petri dish into different sections (usually 2 to 6) and in each section we can scratch different samples using this method. Therefore, it is used in clinical laboratory for culture of urine, sputum, pus, etc. if several samples have arrived at the same time. In this way we can save resources and achieve maximum performance with minimal resources.

4. Radiant stripes

It is another scraping method in which the inoculum is first scraped onto an edge and then spread across the edge in vertical lines. Finally, the vertical lines intersect diagonally. This method is suitable for growing pure cultures and also for diluted samples.

There are other modified forms of stripes such as:

5. Semi-quantitative scraping

Urine culture is routinely performed. It is a modified form of continuous stripes. This method uses a calibrated loop (typically a 1 or 2 µL loop) to withdraw a specified volume of the liquid sample. A filled loop of sample is drawn in a horizontal line down the center of the petri dish and the sample is spread across the entire dish in a single continuous back and forth motion. This method allows us to roughly quantify the viable cargo (in a range, not an exact number) as well as obtain the pure culture immediately.

6. Stripes and zigzags

It is another form of continuous scratching in which a loop of sample is scratched across the board in a single continuous motion in a zigzag pattern. It is usually done to propagate the pure culture and grow it in large numbers.

requirementsScratch-Board-Methode

1. Scratch-Tool

This is a sterile tool used to scrape the sample from the surface of the culture medium. Tools used for scratching include cotton swabs, inoculation handles (either metal or plastic), toothpicks, and wooden, metal, or plastic sticks/wires. The inoculation loop (nichrome wire loop) is used most frequently.

(In this article we talk about the inoculation loop.)

2. culture sample

Bacterial samples can be in the form of suspension, liquid broth or colonies on solid medium. The sample is collected with an inoculating loop and transferred to the surface of a fresh culture medium to perform the listing.

3. Solid culture media

Specific culture media are used to isolate and differentiate suspected (or specific) bacteria. The culture medium is a solid agar medium that is pre-solidified before use.

4. Bunsen burners and other laboratory equipment

A Bunsen burner is used to sterilize the handle and also create a sterile zone around the flame. Other chemicals, sterilization materials and laboratory equipment are also required.

Procedure or protocol of the strip plate method

The general procedure of the scratch plate method can be summarized as follows:

1. Organize all requirements, don PPE, sterilize work surface, and allow all samples and media to come to room temperature if refrigerated.
2. If the sample is highly concentrated, dilution may be useful to obtain isolated colonies. (But not necessary as the sample is diluted during the scratching process).
3. Flame sterilize the inoculation loop and allow to cool. Select a small portion of the isolated colony. (if the sample is in suspension, take a loop out of the sample)

The vaccination procedure is different depending on the method of sowing, let's consider each type:

dial scoring method

1. Take the Petri dish in your left hand and hold it at a 60° angle.

(If you are left-handed, hold the plate in your right hand)

2. The sample is spread over about 1/4^afrom the center of the Petri dish from the rim to the center of the dish with a quick, steady back and forth motion.

(To make it easier, a beginner can draw 2 diagonally intersecting diameters on the back of the petri dish to divide the medium into 4 equal sections)

3. Flame the loop again and let it cool. Rotate the Petri dish 90° counterclockwise and place the loop on the last strip of the first quadrant. Move the handle back and forth to spread the inoculum over the last half of the strips in the first quadrant in the second empty quadrant.

(Be careful not to move the loop to the dashes in the first half of the first quadrant.)

4. repeat the process(iii)scratching the third quadrant and the fourth quadrant.
5. A similar step can be performed for the fourth quadrant. However, many people prefer to stretch a few strokes (6-7 strokes) wide and touch the second half of the strokes in the third quadrant. Also, some prefer to make the last part in a zigzag shape forming a tail.

(Batchwise, the loop is sterilized after each quadrant is scratched. Continuously, the loop does not need to be burned after each quadrant is scratched. However, this is only preferable when the sample is highly diluted.)

T-Kratzer procedure

1. Take the Petri dish in your left hand and hold it at a 60 degree angle.⁰.(If you are left-handed, hold the plate in your right hand)
2. The sample is divided into approximately 1/3^thirdfrom the center of the Petri dish from the rim to the center of the dish with a quick, steady back and forth motion.

(To make it easier, a beginner can draw a letter "T" on the back of the petri dish to divide the medium into 3 sections.)

3. Flame the loop again and let it cool. Rotate the Petri dish 90 degrees⁰counterclockwise and place the loop on the last strip of the first quadrant. Move the handle back and forth to spread the inoculum over the last half of the strips in the first quadrant in the second empty quadrant.

(Be careful not to move the loop to the dashes in the first half of the first quadrant.)

4. repeat the process(iii)Cross out the third quadrant. As with quadrant hatching, you can follow any of the hatch patterns in all 3^thirddial.

Continuous scratch process

1. Take the Petri dish in your left hand and hold it at a 60 degree angle.⁰.(If you are left-handed, hold the plate in your right hand)
2. Place the handle at one end of the plate and spread the inoculum from that point toward the center of the plate in one continuous motion.
3. Rotate the plate 180⁰and without sterilizing the handle, follow step(ii)to scrape the remaining half of the board.

Radiant scratching method

1. Take the Petri dish in your left hand and hold it at a 60 degree angle.⁰.(If you are left-handed, hold the plate in your right hand)
2. Spread the inoculum in a gentle zigzag motion over the near edge of the agar plate.
3. Sterilize the handle and let it cool. The primary dot line was then extended radially to the very edge of the Petri dish.(Be careful to pull the lines further apart.)
4. Flame the loop again and let it cool. Then draw horizontal lines crossing the radial stripes.

Semi-quantitative scraping method

1. Take the Petri dish in your left hand and hold it at a 60 degree angle.⁰.(If you are left-handed, hold the plate in your right hand)
2. Perform a full loop of the sample (urine) using a calibrated loop.
3. Draw the sample in a vertical or horizontal line (primary line) in the middle of the plate.
4. Using the same loop, spread the inoculum by continuously moving the loop back and forth (zigzag) along the primary row.

5. Label at the bottom of the plate with the date, name, sample identification and other necessary information.
6. Incubate the plate upside down under appropriate incubation conditions (mainly 24 hours at 37⁰C).

Interpretation of the results of the scratch method

• The results can be reported after the incubation period (mainly 24 hours to 37 days).⁰C). After incubation, terminal lines of isolated colonies are observed. The morphologies of isolated colonies are tabulated. If the culture medium used is of the indicator type, changes in the medium (e.g. fermentation of lactose on MacConkey agar) are also tabulated.
• There is a large overgrowth of overgrown colonies in the area of ​​the first hatch and there are gradually fewer colonies in the subsequent hatches, resulting in some well isolated colonies in the last hatch.
• Colonies with a similar appearance are expected in inbred cultures.
• If the sample contains only one species, colonies with similar morphologies will be obtained. However, with mixed cultivation, colonies with different morphologies are obtained.
• If there are different types of colonies, each colony must be reseeded on a different plate to obtain a pure culture of each type.

[exception: In some cases where the characteristics of colonies from two or more species of bacteria are the same, all colonies may appear similar even if they are from a different individual.]

Precautions to be taken during the scratch plate method

1. Media must be properly solidified before use. If chilled, allow to come to room temperature.
2. Before scraping, check the media for water droplets and/or contamination or foreign matter.
3. Follow proper safety protocols. Treat all unknown or clinical specimens as hazardous and follow appropriate safety procedures.
4. If using a toothpick to scratch, use the blunt end by holding the pointed end between your thumb and ring finger at a 10 to 20 degree angle.⁰the average. Throw it away after crossing each quadrant and get a new one to cross the second quadrant.
5. If the sample is in suspension, mix the suspension well before removing the inoculum. Follow aseptic procedures during the process. Burn the rim of the test tube or bottle before and after taking the inoculum. When using a micropipette, do not touch the barrel of the pipette on the side of the tube or vial.
6. If the sample is a colony, gently touch the colony with a cold, sterile pen. Don't take all of the cologne or a large portion, just touch the cologne and it will do.
7. Always work in a sterile area (between the flames of a Bunsen burner or in a biosafety booth). Properly sterilize the inoculation loop before and after use. If flame sterilization is to follow, ensure the handle has cooled before use.
8. Appropriate media must be selected.
9. Use only a small amount of sample. Heavy inoculum does not produce isolated colonies.
10. Spread gently without applying too much pressure.
11. Flame the loop after crossing each quadrant.
12. Rotate the board counter-clockwise after traversing each quadrant. Do not scratch from the first half of the front quadrant.
13. Follow the appropriate stripe pattern. If multiple samples are scratched on the same plate, ensure that there is at least a 20-30 mm gap between the scratch areas on each sample.
14. Label correctly and incubate under appropriate conditions.

Applications of the strip plate method

1. It is used to obtain a pure culture from the mixed culture for morphological, biochemical and molecular testing for identification and other applications.
2. It is used to define the sample as pure or mixed species.
3. It is used to study the properties of bacterial colonies.
4. It is used to produce a colony of genetically identical individuals.
5. It is used in inoculation of clinical specimens in diagnostic laboratories to grow isolated colonies of pathogens.
6. It is used in urine culture to isolate pathogens and semi-quantify uropathogens to determine the significance of an infection. A calibrated loop is used for this.

Advantages of the scratch board method

1. It is a simple, reliable, convenient and easy-to-perform inoculation method.
2. Yields well isolated colonies, each with genetically identical individuals; Therefore, we can do more testing and applications on the isolates. Hence it follows in clinical diagnosis.
3. Dilution is performed in conjunction with the inoculation (or vaccination) process, eliminating the need for separate sample dilution.
4. Allows you to manually control the sample as well as sample size and inoculation area in a petri dish.
5. Different stripe patterns provide flexibility in selecting the appropriate method based on sample size, Petri dish availability, and other requirements.
6. It is a convenient and less time-consuming method of growing aerobic organisms.
7. We can use an example in either state; Broth or suspension, as well as colonies from solid media.

Limitations of the risk plate method

1. It is a qualitative isolation method, therefore it does not help to quantify the microbial load.
2. It is more suitable for aerobic than anaerobic organisms.
3. Syntrophic bacteria cannot be purified with this method.
4. It is not suitable if your sample size is large and you have a very high viable number. If we take a strong inoculum, it is possible that there will be no isolated colonies after incubation.
5. You will need certain pre-consolidated media prior to application. So either we need advance information about likely microorganisms in the sample or we need different types of media.
6. There is a chance that scraping will scratch the agar surface if you are not skilled enough and the medium is freshly prepared.
7. The procedure is time-consuming and requires an additional tool (inoculation loop) for smearing.
8. The chance of contamination during the process is high because we have to open the petri dish lid and use the inoculation loop all the time. Therefore, a sterile field and regular sterilization of the handle must be in place.

references

1. Sanders E. R. (2012). Aseptic Labor Techniques: Seeding Methods.Journal of Viewed Experiments: JoVE, (63), e3064.https://doi.org/10.3791/3064
2. Treaty on Microbiology and Immunology (2012), 2^{Dakota do Norte}Install editions. Subah Chandra Pariya. ISBN: 978-81-312-2810-4
3. Practical handbook of microbiology, 2^{Dakota do Norte}edition. Edited by Emanuel Goldman and Lorrence H. Green. CR press. Taylor & Francis Group. 6000 Broken Sound Parkway NW, Suite 300.
4. VAN Soestbergen, AA & Lee, C.H. (1969). Pour slabs or striped slabs?Applied Microbiology,18(6), 1092–1093.https://doi.org/10.1128/am.18.6.1092-1093.1969
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