Unit 3

Unit 3: Microbial Growth, Aseptic Inoculation & Streak IsolationBy Heather Fitzgerald, Patricia G. Wilber and Karen Bentz, and Andrea Peterson, 2018Creative Commons Attribution-NonCommercial 4.0 International License.

I. IntroductionBacterial GrowthBacterial cells reproduce by binary fission, a cloning process whereby one bacterial cell will divide into two identical cells. If the bacterial cells have ideal growing conditions, their average rate of cell division is twenty minutes.

Figure 3-1. Binary fission of bacteria (blue circles) on a solid growth surface results in a mass of bacterial clones called a colony. Colonies are visible to the naked eye. By the time we see the colonies they consist of millions of clones of the original cell.

Figure created by Heather Fitzgerald and Patricia G. Wilber

Growth MediaGrowth media is most commonly prepared in three different physical forms: liquid (broth), semi-solid (deep), and solid (Petri dish and slant). Since all growth media contain nutrients, the major difference in these three forms is the amount of solidifying agent, agar, added to each mix. The liquid broth lacks agar, the semi-solid deep has less than 1% agar and the solid Petri dish and slant have more than 1% agar.

Bacteria require nutrients and water for growth. Two common sources of nutrients in bacterial media are digested milk and soy, but digests of beef and yeast are also used. Chemicals which balance the pH and salt concentrations are also generally added. Agar is extracted from a red marine algae, and is used to solidify media because most bacteria cannot digest agar. Thus it provides a surface for bacterial growth that will not turn to mush. Agar is also useful for growing bacteria because it is solid at room temperature (23oC), it remains solid when the bacteria are incubated at 37 oC, but it becomes a liquid at 100oC and thus can be dissolved in media and poured into tubes and Petri plates.

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Table 3-1. Physical characteristics and forms of common growth media.

Figure created by Patricia G. Wilber

Aseptic Transfer and Inoculation TechniquesIt is important when transferring or moving bacteria from one location (or growth medium) to another to use aseptic technique. Aseptic means without contamination (‘a’ = without and ‘septic’ = contamination. By using aseptic technique, we will (hopefully) grow only the bacteria we are studying, without contamination by other living organisms.

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Inoculation is transfer of a bacterial sample onto a growth media for the purpose of growing the sample. Of course, you must use aseptic technique to avoid contamination.Tools used for inoculation must be sterile. That is, there is nothing growing or alive on the tools. Tools that will be used in lab include metal “needles” and loops, cotton swabs, and wooden dowels.

Metal and glass tools must be sterilized for 20-30 seconds with the micro-incinerator, prior to use.

Swabs and wooden dowels can be used once and then must be disposed of in the sharps container.

Figure 3-2. Metal needle (A) and metal loop (B). Sterilize with the micro-incinerator (C) for 15-20 seconds prior to use. DO NOT REST THE TOOL IN THE INCINERATOR. ALWAYS HOLD IT.

Figures created by Patricia G. Wilber; photograph by Heather Fitzgerald

LabelingUse a Sharpie to label all the media that you will inoculate with the following:

Your name or initials Today’s date Medium type (e.g. T-soy) Organism you will inoculate onto the medium e.g. Escherichia coli (you can use

abbreviations like Ec so long as you record what Ec means in your notes!).

Label for Test Tubes: Can go anywhere EXCEPT the lid.Label for Petri Dishes: Goes on the edges of the agar side of the dish, NOT the lid. Lids can fall off or move or get lost.

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Figure 3-3. Labeling a Petri dish.

Figure Created by Patricia G. Wilber

Culture Incubation and Disposal Incubation and storage: Your inoculated media is placed in an incubator for 24 hours at 37oC. Many of the organisms we work with in lab are found in or on the human body and therefore prefer body temperature. After incubation your inoculated media is stored in the refrigerator until you return for the next lab. There will be a place your instructor will designate for collection of media to be incubated.

Disposal of cultures for decontamination: When you have finished observing your bacterial results, it is important to dispose of your bacteria in the proper place so they can be decontaminated properly. Remember that this lab uses BSL-1 and BSL-2 level human pathogens. Dumping these bacteria into the wrong disposal area can result in infection, sickness and possibly death of others. Briefly:

Used Petri dishes: are disposed of in the big floor buckets that are lined with a red biohazard waste bag.

Used Glass test tubes: Are disposed of in the orange wire test-tube racks on the side counter.

Scientific NamesBe sure to write the names of your bacteria using proper scientific nomenclature on quizzes and papers. All of the following are acceptable:

Staphylococcus aureus (underline genus and species names separately) STAPHYLOCOCCUS AUREUS (all capital letters) Staphylococcus aureus (italicized genus and species) ONLY USE THIS IF YOUR ARE

TYPING. Do not try to write in italics.

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II. DAY ONE Introduction to Inoculation Techniques

Sterilization of an Inoculating Tool using a Micro-incinerator https://youtu.be/6xFWIqU0uXg

Sterilization of an Inoculating Tool using a Bunsen Burner https://youtu.be/e8j40SZHmlQ

Inoculating a Petri Plate with the Streak Isolation Technique https://youtu.be/hNnzype-pbY

What a good streak isolation looks like https://www.youtube.com/watch?v=jLcjd-45yQM

Transferring organism from a Slant Medium to a Broth medium https://youtu.be/-_GwZZ7Yrj4

Transferring organism from a Petri dish to a Slant https://youtu.be/xkX4H15cvdM Transferring organism from a Broth to a Deep https://youtu.be/y0igQ7mfD_k Creating a Lawn Petri Plate (Environmental Sample Example)

https://youtu.be/9F4CUV5JW-UVideos by Corrie Andries and Karen Bentz

Materials for Entire Lab Metal Inoculating loops, needles and cotton swabs Test tube racks to hold tools and tubes Micro-incinerators Black Sharpie-style markers Appropriate personal protective gear (lab coats, gloves, face shield, hair ties) Media (For each student)

o 1 T-soy broth in test tubes (T-soy broth)o 1 T-soy agar as a semi-solid deep in test tubes (T-soy deep)o 1 T-soy agar as a solid poured as a slant (T-soy slant)o 1 T-soy agar plate, solid (T-soy plate) (for the environmental sample)o 1 Chocolate agar plate (for the streak isolation)

Bacteria Cultures, from which to inoculate new media (Note: substitutions may be made as needed by lab technicians)

o Escherichia coli (Ec) (grown on a T-soy slant)o Pseudomonas aeruginosa (Pa) (grown in a T-soy broth)o Staphylococcus aureus (Sa) (grown on a T-soy plate)

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ProceduresA. Streak Isolation Technique: Isolating Individual Microbe Colonies IntroductionOne of the most important techniques in microbiology is the Streak Isolation Technique. This procedure allows isolation of single bacterial cells that will then grow in to single colonies. This technique can be used so colony morphology can be studied, and it can also allow separation of species from a mixed culture so that the different species can be identified.

To perform this technique, we drag the microbe sample (that contains billions of cells) across an agar surface (usually a Petri dish) in a sequential pattern that reduces the number of cells and allows isolation of individual cells. We won’t be able see these single cells when performing the technique, but we will see the colonies that grow from those isolated cells (that underwent binary fission—See Figure 3-1) on the Petri dish after incubation.

Materials (Per student) for Streak Isolation Inoculating metal loops Test tube racks to hold tools Micro-incinerators Black Sharpie-style markers Appropriate personal protective gear (lab coats, gloves, face shield, hair ties) Medium

o One Chocolate agar Petri plate Bacterial cultures

o Pseudomonas aeruginosa (grown in a T-soy broth)o Staphylococcus aureus (grown on a T-soy plate)

Streak Isolation methodsChoose ONE of the two methods described below and follow it carefully to perform a Streak Isolation.

a. Streak Isolation from a broth1. Use Pseudomonas aeruginosa.2. Label the bottom of your Petri plate with your name, the date, the species and the

type of media. 3. Sterilize your loop using the micro-incinerator or Bunsen burner. Allow it to cool for

15-20 seconds.4. Stir the broth sample with your sterile cooled loop to mix the organism throughout

the liquid. 5. Tap your loop on the inside edge of the upper part of the test tube to decrease the

amount of liquid on the loop. YOU DO NOT WANT MUCH AT ALL!!!6. Clam shell your Petri dish. Smear your loop in a vertical 1 cm smear on your plate.

(“A” on Figure 3-4).

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7. Sterilize your loop using the micro-incinerator to get rid of the bacteria—we are trying to get isolation. Allow it to cool for 15-20 seconds

8. Clam shell your Petri dish. Using the edge of the newly sterilized and cooled loop, gently streak through your smear “A”, 10 times, from the outer edge of the plate inwards, as shown on the diagram. This is streak “B”.

9. Sterilize your loop using the micro-incinerator to get rid of the bacteria—we are trying to get isolation. Allow it to cool for 15-20 seconds

10. Using the edge of the cooled loop, gently streak through your smear “B”, 10 times, from the outer edge of the plate inwards. This is streak “C”.

11. Sterilize your loop using the micro-incinerator to get rid of the bacteria—we are trying to get isolation. Allow it to cool for 15 seconds

12. Using the edge of the loop, gently streak through your smear “C”, 10 times, from the outer edge of the plate inwards. This is streak “D”.

13. Sterilize your loop using the micro-incinerator to get rid of the bacteria—we are trying to get isolation. Allow it to cool for 15-20 seconds

14. Streak through “D” and into the middle on the plate WITHOUT touching streak “A”15. Reflame loop.16. Place your inoculated Petri plate upside down (see Fig. 3-5) in a Petri plate rack for

incubation.

Figure 3-4. Streak Isolation inoculation pattern and the clam shell technique for aseptic removal of bacteria from a Petri dish.

Figures by Patricia G. Wilber

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b. Streak Isolation from a plate (Petri dish)1. Use Staphylococcus aureus.2. Label your plate with your name, the date, the species and the type of media. 3. Using a sterile, cooled loop, obtain 1-2 colonies from the source bacteria grown on

the Petri plate (that means NOT VERY MUCH) of identical morphology (shape) using just the tip of your loop. USE A VERY SMALL AMOUNT!! THE SIZE OF A SMALL FRECKLE! Smear those colonies in a vertical 1 cm smear on your plate with the tip of your loop (“A” on Figure 3-4).

4. Follow steps 6-16 above.

Precautions Make sure to cool the metal inoculation loop before transferring bacteria to the Petri

plate for the first streak (A), otherwise you will kill your bacteria. Use VERY SMALL amounts of bacteria when sampling from a plate. A sample that is

1mm X 1mm has billions of bacteria. Make sure to sterilize the metal loop after the original (A) streak and each subsequent

streak. The idea is to REDUCE the number of cells each streak. Don’t gouge the surface of the agar.

Figure 3-5. Proper incubation for Petri dishes: AGAR SIDE UP. Agar side up allows you to read the label and reduces condensation in the plate.

Figure created by Patricia G. Wilber

B. The Lawn Technique: Microbes in the EnvironmentIntroductionAs a first introduction to the world of unseen microbes that are part of your everyday lives, you will sample and try to grow microbes from the laboratory environment. Samples can be taken from anywhere in the lab but please don’t sample directly from humans or human fluid samples. These are more likely to contain pathogenic organisms!

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Materials (per student) One sterile, wrapped, cotton swab One tube sterile water Black Sharpie-style marker Medium

o One T-soy agar plate Bacterial cultures

o Microbes to be sampled from the classroom environment (shoe!)

Method for the environmental sample1. Label the Petri plate appropriately, including the sampling location.2. Wet the sterile cotton swab with the sterile water. 3. Swab something in the classroom environment. The bottom of your shoe will usually

produce amazing results!4. Clam-shell the T-soy agar Petri plate, and create a lawn on the surface of the agar.

To create a lawn, draw your swab across the surface of the agar in the left to right pattern shown in Figure 3-6. Make sure you cover the whole area, leaving no gaps between the swab lines, but do leave a bit of the edge visible. Now make a similar pattern but up and down. The idea is to create bacterial growth in a solid pattern over your plate.

5. Discard the cotton swab in the sharps waste bucket. Do not reuse the swab!6. Dispose of the water that was previously sterile in the biohazard tube rack.7. Place the Petri dish, inverted (See Figure 3-5), in the rack for incubation.

Figure 3-6. Using a swab to make a lawn on a petri plate.

Figure created by Patricia G. Wilber

Precautions Do not sample from human skin, orifices or clothes Do not sample from human fluids, or bathrooms Make sure to run the swab over the sample site several times.

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C. Inoculating from a Slant (solid) into a Broth (liquid)

Figure 3-7: Inoculating from a Slant to a Broth.

Figures created by Patricia G. Wilber

Method for inoculating from a slant to a broth1. Properly label your uninoculated broth medium.2. Sterilize your loop and store in the test tube rack.3. Pick up your un-inoculated broth and your E. coli slant. 4. Unscrew the lids and place them with the open end facing down, on a clean Kimwipe to

prevent bacteria from landing on the inside. DO NOT MIX THEM UP.5. Pick up your cooled, sterilized loop. 6. The bacteria will resemble a whitish slime on the slanted surface of the agar.

GENTLY draw the loop over the surface of the slant to pick up a very small amount of bacteria- the area of a small freckle (about 1mm X 1mm) of the E.coli. A sample of this size will contain millions of bacteria cells. Do NOT dig into the agar.

7. Put the loop with the E. coli in the broth and gently swirl the loop. Remove the loop but keep holding it.

8. Put the lid back on the E. coli slant. 9. Put the lid back on your newly inoculated T-soy broth.10. Leave the lid a bit loose on your broth so that the bacteria have access to oxygen. 11. Re-sterilize your metal tool and return to the canister.12. Place your newly inoculated T-soy broth in the test tube rack designated for incubation.

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D. Inoculating from a Broth (liquid) to a Deep (Semi-solid)

Figure 3-8: Inoculating from a Broth to a Deep.

Figure created by Patricia G. Wilber

Method for inoculating from a broth to a deep 1. Label your medium.2. Incinerate your needle and store in the test tube rack.3. Pick up your un-inoculated deep and your Pseudomonas aeruginosa broth. 4. Unscrew the lids and place them with the open end facing down, on a clean kimwipe.

DO NOT MIX THEM UP.5. Pick up your cooled, sterilized needle. 6. Put the needle into the Pseudomonas aeruginosa broth. 7. Remove the needle from the broth and stab the needle into your solid un-inoculated

deep about ½ to ¾ of the way down. 8. Remove the needle but keep holding it.9. Put the lid back on the Pseudomonas aeruginosa broth.10. Put the lid back on your newly inoculated T-soy deep.11. Leave the lid loose on your deep so that the bacteria have access to oxygen. 12. Re-sterilize your metal tool and return to the canister.13. Place your newly inoculated T-soy deep in the test tube rack for incubation.

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E. Inoculating from a Petri Dish (solid) to a Slant (solid)

Figure 3-9. Inoculation from a petri dish to a slant

Figures created by Patricia G. Wilber

Method for inoculating from a Petri dish to a slant 1. Label your medium.2. Incinerate your loop and store in the test tube rack.3. Unscrew the lid of your un-inoculated slant and place it with the open end facing down

on a clean Kimwipe. 4. Pick up your cooled, sterilized loop. 5. Clam-shell the lid of your Staphlyococcus aureus containing Petri dish and pick up only a

very small amount of bacteria (about 1mm X 1mm). A sample of this size will contain millions of bacteria cells. Do NOT dig into the agar.

6. Replace the lid of the Petri dish.7. Streak the surface of the slant as shown in Figure 3-12. Do NOT dig into the medium.8. Remove the loop but keep holding it.9. Put the lid back on your newly inoculated T-soy slant.10. Leave the lid loose on your slant so that the bacteria have access to oxygen. 11. Re-sterilize your metal tool and return to the canister.12. Place your newly inoculated T-soy slant in the test tube rack for incubation.

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III. DAY TWO RESULTS AND INTERPRETATION A. Streak isolationFigure 3-10. Hoped for results from a streak isolation on the left. The example on the left was created by a student using the Streak Isolation from a Plate technique. The result on the right shows too much bacteria resulted in no isolated colonies.

Photographs by Patricia G. Wilber

Observe your Streak Isolation Petri dish. 1. Did you do your streak isolation from a broth or a plate? (circle one)

2. In the space below, draw or insert a photograph of the results from your isolation.

3. Do you have 8-10 isolated colonies? (yes or no)

4. How could you improve your next streak isolation?

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B. Environmental SampleIf you see colonies of various colors, sizes and shapes on the surface of your Environmental T-Soy Petri plate, you have successfully cultivated microbes from the environment you sampled. Each species forms a unique colony that has its own combination of size, appearance and color.

(1) The size of the colony – commonly ranging from 0.5mm to 5mm in diameter. Measure with a ruler, but do not touch the colony.

(2) The appearance of the colony:o smooth (a shiny or reflective surface)o rough (a matte, granular, or bumpy surface) o mucoid or viscous (a slimy, gel-like or gummy surface)

(3) The color of the colony:o Usually white, clear or tan, but orange, blue, pink and other colors are possible!

Figure 3-11. Colonies of Sinorhizobium meliloti are approximately 1 to 2 mm in diameter (size). They lack pigmentation and are clear and slightly brown (color). Colonies are smooth and viscous (appearance), and are circular in form.

http://www.microbelibrary.org/component/resource/laboratory-test/3136-colony-morphology-protocol Authors Donald Breakwell, Christopher Woolverton, Bryan MacDonald, Kyle Smith, Richard Robison. Accessed 5/26/2015, Creative Commons Attribution – Noncommercial – NoDerivatives 4.0 International

1. Based on the various different colony characteristics you can see, estimate how many different species you have on your environmental plate. Remember that each unique type of colony represents one species. Circle the number of your estimate.

a. 1-5b. 6-10

c. 11-20d. More than 20

2. List three features you focused on to differentiate between the colonies.

SAVE your plates as indicated by your instructor for staining procedure in the next Unit!

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C. Broth For your broth, the medium may be cloudy with bacteria, or bacteria may have settled on the bottom of the tube.If you do not observe growth, talk to your instructor.

D. DeepFor your deep you should see growth where you stabbed into the medium. If you do not observe growth, talk to your instructor.

E. SlantHopefully, you will see bacterial growth in a streak on the surface of your slant. The growth will probably be white, clear or tan. If you do not observe growth, talk to your instructor.

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IV. Post-Activity Questions Name: ______________________

1. Explain the role of agar in media.

2. Draw and identify a slant, a deep, a broth and petri dish. Next to each draw and name the tool that should be used for inoculation.

3. Write any scientific name in proper format for

a. a quiz where you have to hand write the name.

b. a paper where you can type the name.

4. Match the following terms with the descriptions:Term Descriptiona. deep To place newly inoculated media in an environment optimal for growth.b. Sterile A procedure that is done without contamination.c. media One form of semisolid media.d. slant Is made with materials rich in vitamins and nutrients.e. Incubate One form of solid media.f. aseptic A term applied to metal tools after they are heated

5. Angel inoculated a Petri dish with E. coli. When she looks at her plate after it has been incubated for 24 hours, she observes that the plate looks exactly the same as it did on the day of inoculation.

a. This indicates there was no ________________ on her plate.

b. List three things that may have occurred that would give her this result.

6. Why should you keep test tube lids on your inoculated cultures for incubation screwed on loosely?

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7. The sink and door knobs typically have far fewer species of bacteria than the bottom of a shoe. Develop a hypothesis for the sink and for the door knob as a possible explanation for these results.

a. Sink vs. shoe

b. Door knob vs. shoe

8. Suppose you have a mix of two bacteria in a culture, bacteria “A” and bacteria “B”. You inoculate a sample containing equal numbers of these two bacteria types using a streak isolation technique onto a plate. Draw your expected results below (using colored pencils or the computer draw tools and labels).

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