ExerciseⅡ Basic Manipulation of Microorganism（Ster...
1.Culture Transfer Instruments,Techniques,and Isolation
Materials
inoculating loop
noculating needle
Bunsen burner
24-hour tryptic soy broth and tryptic soy agar
tryptic soy broth tubes
tryptic soy agar slants
tryptic soy agar deeps
Learning Objectives
Each student should be able to：
（1）Correctly use an inoculating loop and needle.
（2）Use aseptic technique to remove and transfer bacteria for subculturing.
（3）Understand the reasoning behind pure culture preparations.
Principles for Culture Transfer Instruments and Techniques
Inoculating needles and loops[Figure 1（a），（b）]are used to aseptically transfer microorganisms from broth，slant，or agar cultures to other media.Both may consist of handles，a shaft，and a turret，which holds a nickel chromium or platinum wire.If the wire is straight，it is an inoculating needle；if a loop is present，it is an inoculating loop.Before using either，the end of the wire must be sterilized by passing it slowly through the tip of the flame from a Bunsen burner or into a Bacti-Cinerator（Figure 2）.When done correctly，all parts of the wire will turn red with heat.The needle or loop should then be used before it becomes contaminated.After you have finished using an inoculating loop or needle，it should be thoroughly flame-sterilized.
Microorganisms are transferred from one culture medium to another by subculturing，using specific procedures and aseptic technique.Asepsis means free from sepsis a toxic condition resulting from the presence of microorganisms.This aseptic technique is of such importance that it will be used in most of the exercises in this manual.Since microorganisms are always present in the laboratory，if aseptic technique is not followed，there is a good possibility that external contamination will result and will interfere with the results.Proper aseptic technique also protects the laboratory worker from contamination with the culture.
Figure 1 Microbiological Transfer Instruments
（a）Inoculating needle（b）inoculating loop
Principles for Isolation of Pure Cultures and Their Maintenance
Once discrete，well-separated colonies develop on the surface of the streak plate，selected ones may be picked up with an inoculating needle and transferred to separate culture tubes，such as tryptic soy agar slants（the type of agar will depend on the microorganism）.Where possible，bacteria from the center of a colony are transferred，because the center is less likely to be contaminated than the edges.Each slant now represents the growth of a single species of microorganism and is called a pure or stock culture.
One way in which many cultures may be maintained for relatively long periods is by sealing them with sterile mineral oil in order to prevent moisture loss.
Procedure for Culture Transfer Instruments and Techniques
Aseptic Technique
（1）Using a wax pencil，label the tube or plate to be inoculated with the date，your name，and the name of the test microorganism[Figure 2（a）].
（2）Gently mix the primary culture tube in order to put the bacteria into a uniform suspension[Figure 2（b）].The tube can be tapped to create a vortex that will suspend the microorganisms，or if a vortex mixer is available，it can be used.
（3）Place the stock culture tube and the tube to be inoculated in the palm of one hand and secure with the thumb.The tubes are then separated to form a V in the hand[Figure 2（c）].They should be held at an angle so that the open ends are not vertical and directly exposed to airborne laboratory contaminants.
（4）Using the other hand，flame the inoculating loop or needle over a Bunsen burner until the wire becomes red-hot[Figure 2（d）].
（5）Using the same hand that is holding the inoculating loop，remove the caps from the two tubes，hold them between your fingers，and briefly flame the necks of the tubes over a Bunsen burner[Figure 2（e）]by passing them through the flame.
However，do not allow the tubes to become red-hot.
（6）Cool the hot loop in the broth culture until it stops“hissing.”With the sterile inoculating loop，transfer 1 drop of culture from the stock culture tube into the new broth tube.At this point，one could also transfer to a glass slide，streak the surface of a slant，or streak the bacteria onto the surface of a petri plate[Figure 2（f）].When picking up bacteria from a slant，cool the hot loop or needle by holding it against the top of the slant until it stops“hissing”.
（7）Reflame the neck of the tubes[Figure 2（g）].
（8）Recap the tubes[Figure 2（h）].
（9）Reflame or sterilize the loop or needle[Figure 2（i）].
Figure 2 Aseptic Technique for Bacterial Removal and Subculturing
Using aseptic technique,perform the following transfers:
1）With the inoculating loop，transfer the S.marcescens tryptic soy broth culture to a tryptic soy agar slant.Also，inoculate a tryptic soy broth tube with S.marcescens，using the inoculating loop.
2）With the inoculating needle，transfer the S.marcescens to a tryptic soy agar deep tube.
This is done by plunging the inoculating needle of S.marcescens into the tube without touching the walls of the tube.Penetrate the medium to i of its depth.The inoculating needle is then withdrawn from the tube[Figure 3（a）-（c）].
3）Using the inoculating loop，make a slant-to slant transfer.This is done by gently streaking the surface of the slant in the form of a serpentine（wiggly or Sshaped）line[Figure 3（d）].If there is liquid at the base of the slant，the tube may be tilted after inoculation so that the liquid runs over the slant surface.This will moisten the surface and spread out the bacteria.
4）Place the tubes in a test-tube rack or a clean vegetable can and incubate at 35℃for 24 to 48 hours.Afterwards，examine all of the tubes for the presence of bacterial growth.
Figure 3 Transferring Techniques
（a）-（c）Stab technique for transferring bacteria.Notice that the inoculating needle is moved into the tube without touching the walls of the tube，and the needle penetrates medium to i its depth.（d）Technique for streaking the surface of a slant with a loop.
2.Spread-Plate and the Streak-Plate Technique
Materials
24 to 48 hours tryptic soy broth cultures of Escherichia coli（white colonies），Serratia marcescens（red colonies；Micrococcus roseus can also be used），and Bacillussubtilis（white or cream colonies）.
Bunsen burner
inoculating loop
95%ethyl alcohol(https://www.daowen.com)
L-shaped glass rod
wax pencil
500 ml beaker
3 tryptic soy agar plates rulers
Tryptic soy agar pours
boiling water bath
48℃to 50℃water bath
petri plates
inoculating loop
Learning Objectives
Each student should be able to
1）Understand the purpose of the spread-plate technique.
2）Perform the spread-plate technique.
3）Understand the purpose of the streak-plate technique and differential media.
4）Perform a streak-plate technique and isolate discrete colonies for subculturing.
Principles of Spread-Plate Technique
In natural habitats，bacteria usually grow together in populations containing a number of species.In order to adequately study and characterize an individual bacterial species，one needs a pure culture.The spread plate technique is an easy，direct way of achieving this result.In this technique，a small volume of dilute bacterial mixture containing 100 to 200 cells or less is transferred to the center of an agar plate and is spread evenly over the surface with a sterile，L-shaped glass rod.The glass rod is normally sterilized by dipping in alcohol and flamed to burn off the alcohol.After incubation，some of the dispersed cells develop into isolated colonies.A colony is a large number of bacterial cells on solid medium，which is visible to the naked eye as a discrete entity.In this procedure，one assumes that a colony is derived from one cell and therefore represents a clone of a pure culture.
After incubation，the general form of the colony and the shape of the edge or margin can be determined by looking down at the top of the colony.The nature of the colony elevation is apparent when viewed from the side as the plate is held at eye level.
Principles of the Streak-Plate Technique
Isolated，pure colonies can also be obtained by the streak-plate technique.In this technique，the bacterial mixture is transferred to the edge of an agar plate with an inoculating loop and then streaked out over the surface in one of several patterns.At some point on the streaks，individual cells will be removed from the loop as it glides along the agar surface and will give rise to separate colonies（Figure 1）.Again，one assumes that one colony comes from one cell.The key principle of this method is that by streaking，a dilution gradient is established on the surface of the plate as cells are deposited on the agar surface.Because of this gradient，on fluent growth occurs on part of the plate where the cells are not sufficiently separated，and individual，well isolated colonies develop in other regions of the plate where few enough cells are deposited to form separate colonies that can be seen with the naked eye.Cells from the new colony can then be picked up with an inoculating needle and transferred to an agar slant or other suitable medium for maintenance of the pure culture.
Figure 1 Streak Plate
Notice the well-isolated colonies of E.coli（white）and S.marcescens（red）Corresponding book color chart NO.1
Procedure of Spread-Plate Technique
（1）With a wax pencil，label the bottom of the agar medium plates with the name of the bacterium to be inoculated，your name，and date.Three plates are to be inoculated：（a）one with B.subtilis，（b）one with S.marcescens，and（c）one with the mixture.
（2）Pipette 0.1 ml of the respective bacterial culture onto the center of a tryptic agar plate[Figure 2（a）].
（3）Dip the L-shaped glass rod into a beaker of ethanol[Figure 2（b）]and then tap the rod on the side of the beaker to remove any excess ethanol.
（4）Briefly pass the ethanol-soaked spreader through the flame to burn off the alcohol[Figure 2（c）]，and allow it to cool inside the lid of a sterile petri plate.
（5）Spread the bacterial sample evenly over the agar surface with the sterilized spreader[Figure 2（d）]，making sure the entire surface of the plate has been covered.Also make sure you do not touch the edge of the plate.
（6）Immerse the spreader in ethanol，tap on the side of the beaker to remove any excess ethanol，and reflame.
（7）Repeat the procedure to inoculate the remaining two plates.
（8）Invert the plates and incubate for 24 to 48 hours at room temperature or 30℃.
（9）After incubation，measure some representative colonies and carefully observe their morphology.Record your results in the report.
Procedure of Streak-Plate Technique
1.Melt three sterile，capped tubes of tryptic soy agar by heating them in a boiling water bath until melted[Figure 2（a），（b）].
Figure 2 Spread-Plate Technique.
2.Cool the tubes in a 48℃to 50℃water bath for 10-15 minutes.
3.Remove the cap，flame the lip of the tube，and pour the agar into a petri plate[see figure 2（c）-（f）].Be careful to keep the lid of the plate covering the bottom and the mouth of the tube while pouring the agar.Do the same for the other two plates.
4.After pouring the plates，allow them to cool for a few minutes on the bench top.With a wax pencil，mark on the bottom of the plate the name of the bacterium to be inoculated，your name，and date.
5.Aseptically remove a loopful of the bacterial mixture[Figure 2（a）].
6.Streak out the loopful of bacteria on the agar plate that you have prepared as follows：
1）Carefully lift the top of the petri plate just enough to insert your inoculating loop easily[Figure 3（a）].The top should cover the agar surface as completely as possible at all timesin order to avoid contamination.Insert the inoculating loopful of bacteria and spread it over a small area（area 1）at one edge of the plate as shown in Figure 3（b）in order to make effective use of the agar surface.This is accomplished by letting the loop rest gently on the surface of the agar and then moving it across the surface each time without digging into the agar.
2）Remove the inoculating loop and kill any remaining bacteria by flaming them.Then insert the loop under the lid and cool it at the edge of the agar near area 1.
3）Rotate the plate while carefully keeping in mind where the initial streaks ended（use the marked quadrants as a guide）and cross over the streaks in area 1[Figure 3（b）].Streak out any bacteria picked up as shown in area 2.
4）Remove the loop，flame it，cool in the agar as before，and repeat the streaking process[Figure 3（b），area（3）].
5）If necessary，you can repeat this sequence once more to make a fourth set of streaks（area 4）.Use fewer cross-streaks here than in the previous quadrant.
6）Repeat the above procedure（a-e）for the other two bacteria on two new petri plates.
7）Incubate the plates at 30℃to 37℃for 24 to 48 hours in an inverted position.Afterwards，examine each of the agar plates to determine the distribution and amount of growth in the three or four streaked areas and record your results in the report.
Figure 3 Preparation of a Streak Plate
Arrows indicate motion of the loop.In b，flame and cool the loop between 1 and 2，2 and 3，and 3 and the end of the streak.The goal is to thin the numbers of bacteria growing in each successive area of the plate as it is rotated and streaked so that well isolated colonies will appear in quadrant 3.