Make a Loop and Streak Agar Plates to Isolate Pure Strains of Bacteria

The inoculating loop is the microbiologist's main tool.
Remember to sterilize everything before each use.

Microbiologists use their inoculating loop constantly because it is so easy to sterilize and reuse.This page tells you how to make, sterilize, and use a loop to isolate pure cultures of bacteria. We will also discuss how to inoculate stabs, deeps, slants, and assay medium.

A Short Introduction

You will need some way to transfer bacteria from a culture to fresh medium. A wire which you have heated in the flame of a candle, bunsen burner, or gas kitchen stove flame, or dipped in rubbing alcohol will work nicely. Sterile sticks are sometimes more convenient. If you smear the bacteria very thinly on a sterile potato slice or petri dish of an agar medium, you can cause a single bacterium to grow into a colony after a day or two and that is a pure culture of a single strain of bacteria. If you the restreak that colony, you can make sure it is pure (grew from on bacterium). Then use your sterile loop (wire or stick) to transfer some of a pure colony into a tube or bottle of fresh medium so that you have a pure culture also called a stock culture. It is easy and simple to start a culture collection containing a couple tubes of each bacterium that you have isolated or gotten from other bacteriologists. Your knowledge of bacteria will expand dramatically when you have a small culture collection and compare their characteristics growing on milk, cornstarch, meat broth and other media.

Caution: Often the bacteria will be on tiny bits of meat, soil, or invertebrates. This will produce a colony containing many kinds of bacteria. Therefore you must always restreak one or more times until you get a colony which produces only identical colonies when restreaked. I have never had to restreak more than once.

How to Make an Inoculating Needle or Loop

An inoculating needle is a piece of stainless steel, nichrome, platinum, or other wire mounted in a handle. The wire can be any size. Small diameter wires require less time to heat and cool. Most people use 26 guage platinum or 25 guage nichrome wire. I use 20 guage nichrome because that is the size I have. A 20 guage wire has a larger diameter than a 26 guage wire and takes longer to heat and cool..

To make a loop, bend the tip of the wire into a small circle having an inside diameter of 1 mm or whatever size you want. When you dip the loop into a liquid, it should retain a droplet of water. The loop shown on the left will not hold a droplet of liquid bacteria culture because the open section of the loop is too wide. The tip of the loop must bend around and touch the metal or it will not hold water. With bacteriophage one can calculate the volume of liquid held by the loop see b025a.htm for an example. The wire may be of any convenient length. I cut my wires 13 cm long and that gives enough for the loop and a remainder to reach into 125 culture tubes without handle entering the tube. Most people use shorter wires and allow some of the non-sterile handle to enter the tube. My long wires are harder to handle but I prefer to have only sterile wire inside my stock culture tubes.

If you have a dissecting needle holder, loosen the vise nut, remove the dissecting needle and insert your inoculating needle or inoculating loop. I often make a handle from a 15 cm length of aluminum wire 4 or 5 mm in diameter. I drill a small hole about 1 cm into the handle at one end. Then I hammer on the sides of the handle so the hole closes around the wire. Before inserting the wire I bend about 2 mm of the wire back on its self. A needle I made that way is still nearly as good as new after 18 years of use. You may use a file to round the edges of the aluminum handle. At Salem High School, Indiana, students in metal shop made similar handles from brass brazing rods using the above directions..

My Fungus Hoe: I found some 18 guage stainless steel wire and hammered one end flat and then bent the tip up about 1 mm a little more than 90o and sharpened it like a garden hoe as shown in the figure on the left. I use this to transfer fungi (molds) which grow as tough mats which can't be easily torn loose with a loop. I puncture the mat with my sterile fungus hoe and pull. A bit of the mat comes free and makes an excellent inoculum. Precision tools like these speed my work and make tough jobs easy. Such tools will dress up your science fair entry and show the judge you are the top entry and not just some one who entered because he had to. Bring a magnifying glass so the judge can see your precision "mini hoe". This mini hoe is very good for transferring Streptomycetes, a genus of bacteria that grows as very hard mats.

Using an Inoculating Loop or Needle

You do not have to have both a needle and a loop. If you only have one, the loop is more useful. I dislike inoculating needles because they are more dangerous. I do not have a needle. It is possible to injure and infect an eye with an inoculating needle. Loops are safer. Needles, a straight wire, are mainly used for stabbing bacteria vertically into deep tube of gelatin to determine the shape of the hole digested by the bacteria. I use my loop for such purposes and it is almost as good. I suggest beginners never own an inoculating needle because of the risk of stabbing type injuries to small childern or failure to see them in a darkened room.

Another safety note. Plunge your loop into some sterile agar. Now hold the loop into a hot bunsen burner flame and notice the mini explosion as bits of flaming agar fly every direction. Some of those tiny particles may stay suspended in the air and if they contain living pathogenic bacteria they might infect a person inhaling the dust. Now repeat the experiment but this time hold the loop containing agar above the flame so that the agar is gently dried, then lower the loop into the flame so that the agar burns without throwing particles. This risk of throwing infectious dust into the air is one reason beginners should never work with pathogenic bacteria.

To sterilize your tool, hold it in the flame until the tip begins to glow and then move some distance up the needle so that any contaminants are killed. The needle does not need to glow bright orange. Now hold the loop until it is cool. You can touch a loop to agar to learn whether it is cool. You can touch a needle to agar to cool it and avoid waiting. Touch the needle to a colony of your pure culture. Do not dig into the colony or into then agar, just touch the visible bacteria gently. You may not see anything but you will have thousands of cells on the loop.

To inoculate a tube of medium, push the loop to the butt of the slant or stab or along the surface you as you desire. Then sterilize the loop before you lay it down it down.

Streaking a Plate to Isolate a Pure Culture

Begin by marking the back of the dish with a number on a piece of tape so that you will know the top of the dish. This dish is labelled #2. You may find handling the inoculating loop is more convenient when you set the register mark at 10:30 rather than noon. Or if you are left handed, try 1:30. I am suggesting it is easier to streak at an angle for the same reason that you place your paper at angle for easier writing. Use the same part of the loop in contact with the agar to avoid getting additional bacteria on the plate. Our goal is place the fewest possible number of bacteria on the plate and spread them as far apart as possible. The plate should contain 15 to 25 ml of agar depending upon your preference. If you are going to incubate above 45C you might consider special precautions to reduce evaporation of the agar. For example, I sometimes place the dish in a plastic bag for incubation after streaking is completed.



We are now ready to begin streaking. Flame the loop, dip into a liquid culture or touch a colony very slightly and zig-zag it back and forth onto the top 1/3 of the plate as shown here. If are careful to keep only one spot of the loop always in contact with the plate, You will notice, after the bacteria have grown, that the first part of the streak produces a dense line of colonies, but fewer colonies grow from the final part of the streak. This diagram shows 5 spreading sweeps. I is better if you place them closer to gether so that you get about 10 lines of bacteria in the top third of the plate.


Now, flame the loop again. Rotate the plate counter clockwise 90 degrees and cross the prior streaks to pick up some bacteria and spread them into the 3rd quaradant (quarter) of the plate. One uses about twice as many zig-zags as the diagram shows.

 Don't allow the loop to penetate into the agar as that will yield a clump of bacteria.

I always flame the loop between each quadrant, but many professionals do not. Not flaming the two extra times per plate saves time. Try it both ways and see which method works best for you.       


Flame the loop again. Rotate the plate counter clockwise another 90 degress. touch the agar and cross the prior streaks as before and streak the bacteria on the 4th quadrant of the plate.

Streaking a plate to get lots of nicely separated colonies is not easy. Most beginners fail the first time.

The big secret is to place as few bacteria as possible. If you got only a dozen bacteria on the plate, that would be wonderful, but impossible.

      


Incubate the plate at a permissive temperature for the organism and after a day or two the plate may look somewhat like this. This is an extremely poor diagram of what you are likely to find. This diagram shows a better separation of colonies than you are likely to obtain. It also shows an unrealistic large number of well separated colonies in gradrant 4.

     

      


Use Cover of Dish as a Dust Shield

Comtamination is greatly reduced during streaking when you use the cover of the petri dish as a dust shield as illustrated on the left. Lower the cover so the dish is closed while you are flaming your loop and rotating the dish for the next stage of streaking. Contamination by dust is also reduced if you wipe the table with a damp cloth. The damp table top traps some of the dust. Turn fans off and close windows so that dust is not blown into the open dish. Placing the dish in a paper bag during incubation will allow air to the dish but prevent drafts from blowing dust into the closed dish. Caution: bellows action of the bag can pump dust into the dish.

Pour Plate Method for Isolating Bacteria

The pour plate method is not widely used but may be more convenient in some instances and some experienced workers prefer the pour plate method for isolating bacteria. Melted agar 45 to 50o C is used.

Reasons you might want to try the pour plate method.

Pour plate method seldom yields well separated colonies unless several dilutions are plated. Here is an easy method which gives good results.

Usually one of the 3 plates will produce well separated colonies. If an interesting colony is found but it is not well separated, just sample it with the loop and repeat as above and make 3 new plates.

Spread Plate Method

This method is often used for counting bacteria, but can be used for isolating bacteria. You will need a spreader which is usually made by heating a glass rod and bending into an L or 7 shape.

Caution: If the agar plate is a few hours old and has tiny invisible colonies of bacteria due to contamination, these will be spread over the plate causing errors or failure.

Sterile Wooden Items

Place the wooden items in culture tubes or bottles, then cap or plug, and autoclve. Store the sterile tubes of tools in dust-free boxes.

Sterile Wooden Tooth Picks

Sterile wooden items are widely used by microbiologists because they are safe and speed up the work. Sterile tooth picks are the most widely used. Just put some tooth picks in a bottle or culture tube, replace the cap or use cotton to plug the mouth of the container, then autoclave. To use them, just tilt the tube downward slightly while tapping on the tube so the sticks slide to the tube opening so you can pick up the top toothpick without getting the others contaminated. Touch the sterile end of the toothpick to the sample and then stab or streak the sterile medium. If you have a big project, you can use thousands of these sticks per day.

Sterile Wooden Applicator Sticks

You may wrap with cotton to make swab, but bare stick is very useful and allows you to reach into test tubes when toothpicks are too short. You may use twigs from trees or splinters of wood just as readily.

Some examples: you could stab a rotten apple after you have swabbed the surface with alcohol to sterilize the surface. Then stab the stick into the agar in a petri plate or bottle to start a culture. However, such a stab may contain more than one organism. If you carefully move the tip of the toothpick across and agar surface you can later examine to see if it appears that only one organism was present. Sometimes, I use sterile forceps (tweezers, dipped in alcohol) to remove the toothpick from the supply tube and then after stabbing the rotted fruit, I drop the whole toothpick into a sterile tube of water. Then I agitate the tube to disperse the bacteria off the toothpick. Then I remove the toothpick with sterile forceps and discard. Now I have a dilute suspension of bacteria which I can plate or use as desired.

Some Methods for Isolating Anaerobic Bacteria

Anaerobic bacteria can't be isolated via the above methods because they won't grow in the presence of air. Some are killed by momentary exposure to air. Visit our beginners page on Anaerobic Bacteria for more details.

How to inoculate slants, stabs, deeps, slants, TSI

Slants: Since most of the bacteria beginners work with are aerobes, slants (called slopes in Britian) are best. Cool sterile agar in test tubes at an angle so the agar has a large surface for growing bacteria. Touch your loop to an isolated colony and make a gentle stroke on the surface. Would get a single line of bacteria growing. Food and wastes will diffuse and the culture will live many times longer than if you completely covered the surface with bacteria.

Stabs: Some bacteria grow better or live longer within the agar. For these make stabs by filling the tube about 3 cm deep with agar and cool vertically. Inoculate by plunging the loop to the bottom of the tube.

Deeps: Anaerobic bacteria will not grow in the presence of air. Many of these can be grown in the bottoms of tubes containing about 6 cm of agar. The tubes should be freshly autoclaved or reautoclaved. Cool vertically. Plunge a loop of the anaerobe all the way to the bottom of the tube.

Growth is more reliable when you use a pipet to inoculate the culture.

Many bacteria don't use air, but are not harmed by it. The Lactic Acid Bacteria are such organims and it is common to grow them in deeps. Use a loop to transfer them. No need to use pipet. The lactic acid bacteria are easily isolated from fermented milk products and cheeses.

TSI Slants: Triple sugar iron slants are widely used in the identification of intestinal bacteria. They are useless unless inoculated correctly.

The slant on the left was correctly inoculated with Proteus vulgaris. As the bacteria grow the slant will have aerobic growth, but in the butt growth is anaerobic. Some bacteria (facultative anerobes and perhaps anaerobes) will grow in the butt but aerobes won't. Some bacteria growing in the butt will produce hydrogen sulfide with reacts with the iron and blackens the butt of the tube because iron sulfide is black. Some bacteria will produce acid and turn the phenol red (a pH indicator which is yellow in acid) to yellow in the butt of the tube. There is a trace of glucose in TSI medium and all intestinal bacteria can use it. After the glucose is gone the yellow color in the butt will fade away unless the bacterium can use lactose or sucrose which are the other two sugars (triple sugar, remember). The slant will be red or yellow depending upon the products produced by the bacterium in aerobic state. P. vulgaris can ferment sucrose but not lactose.

This example shows the importance of inoculating diagnostic media properly.

Enrichment Cultures

The above methods work for the isolation of bacteria which are predominate in the sample. Bacteria occurring in low numbers will not be seen. One may first inoculate suitable medium and incubate under favorable conditions of temperature, medium, pH, atmosphere, etc to so that the desired organisms outgrow other species. Then you may use that enriched culture for one or more of the isolation methods mentioned here. See page b034 for some introductory enrichment experiments. Eventually, there will be dozens of pages having enrichment info for milk, food, soil, freshwater and other types bacteria.

You may send private e-messages to Dr. Eddleman and he will reply, usually within 24 hours.


First installed January 1999      Revision #5 1999 Sep 29                H2SO4     1022m  90o C
Bacteria Index Page | INDIANA BIOLAB Web Sites | indbio@disknet.com
Written by Harold Eddleman, Ph. D., President, Indiana Biolab, 14045 Huff St., Palmyra IN 47164