Unit 4

Unit 4: Streak Isolation from a Mixed Broth and Simple Stains

By Heather Fitzgerald, Patricia Wilber and Karen Bentz, and Andrea Peterson, 2018

Creative Commons Attribution-NonCommercial 4.0 International License.

I.Introduction

Streak Isolations

Samples from patients usually contain two or more different bacterial species. Thestreak isolationtechnique will separate a sample into individual cells that then grow into unique colonies. A good streak isolation with two species could hopefully look like this plate by Leanna Gutierrez, Bio 2192, Spring 2016. It has two very distinct and isolated colony types!

Figure 4-1 Streak isolation plate by 2192 student Leanna Guteirrez

Simple Stains

Bacterial cells are usually clear in color. To increase the chance of seeing the microbial cells under a microscope, cells are stained with dyes that are attracted to different parts of the cells. Once cells are stained, features such as overall cell size and shape, and cellular arrangements, can be determined. For a review of cell shapes and arrangements please refer to Unit 2 (Microscopes) of this laboratory manual.

Stains are pigment molecules dissolved in a solvent. The simple stains Methylene Blue, Gram’s Crystal Violet, and Safraninareallpositively charged. Bacterial cell wall components (certain polysaccharides and proteins)arenegatively charged. What do you know about opposites? They attract! Thus,the positively charged stains are attracted to the negatively charged bacterial cells you will stain.

Figure 4-2. Idealized view of staining of bacterial cells with Methylene Blue.Unstained, negatively charged, clear bacterial cells plus positively charged Methylene Blue stain yields blue stained cells.

Figure created by Heather Fitzgerald and Patricia G. Wilber

Figure 4-3. Image of bacillus-shaped bacterial cells (species Clostridium septicum) stained with methylene blue.

Accessed 7-9-2015. This image is in the public domain.

Centers for Disease Control and Prevention'sPublic Health Image Library(PHIL), with identification number#14347.

II.Day 1.

A.Streak Isolation using a mixed Broth.

Materials

  • 1 blood plate per student.
  • Bacterial Culture: 0.05ml Proteus vulgaris (Pv) mixed with 5ml of Streptococcus oralis (Sto) in one T-soy broth tube.

Procedure

  1. Refer to Unit 3 to review the streak isolation procedure from a broth. A broth tends to be easier to streak from than a plate.
  2. STIR your mixed broth thoroughly with your sterilized loop before streaking!
  3. Tap your loop to reduce bacteria before performing the streak isolation.
  4. Perform your streak isolation on your blood plate.
  5. Be sure to label your plate correctly.
  6. Incubate this plate in a candle jar.

Figure 4-4. General procedure for the Streak Isolation Technique.

Figure by Patricia G. Wilber

B.Simple Stain Procedure: Methylene Blue

  • Single Specimen Slide
  • Simple Stain Procedure Video

Videos by Corrie Andries and Karen Bentz

Materials (Per student):

  • Wooden dowel
  • Glass slide
  • Methylene Blue stain
  • Distilled water
  • Kimwipes and lens cleaner
  • Microscopes
  • Bacterial Cultures
  • Your environmental sample from previous lab
  • Your streak isolation plate from the previous lab

Procedure:

  1. Usebacteria from your environmental or streak isolation plate.
  2. Touch the flat end of a wooden dowel to one bacterial colony on your plate.

NOTE: Don’t put too much culture on the slide, as a little goes a long way! A little means less than one bacterial colony or about the size of the letter “o” in this sentence.

  1. Tap(do not rub!!) the flat end of the dowel containing bacteria multiple times in the middle of a new, clean glassslide.

Figure 4-4. Putting bacteria on a slide with a dowel

Figure created by Heather Fitzgerald

  1. Dispose of the dowel in the sharps container on your bench.
  2. Go to the sink and place your slide flat on the metal slide holder (or hold it using a clothespin).
  3. Place several drops of Methylene Blue stain on your slidewhere you placed the bacteria, and let it sit for about 30 seconds. You do not need to flood the slide.
  4. Rinse your slide with DI water.
  5. Pat/dab dry very gently using a Kimwipe. Again, pat, DO NOT RUB.
  6. Examine the stained bacteria with your microscope at a TM of 1000X.

Notes:

  • Make sure the slide is dry before viewing or you will transfer dye to the microscope.
  • Be sure your bacteria are facing up/are on the top of the slide!
  • The oil is placed directly on top of your stained sample. You will not use a coverslip.
  1. Make a drawing or photograph of your bacteria, noting the shape of the cells and arrangement of the cells using correct class terminology. Draw this in Figure 4-4.
  2. Estimate the size of your bacteria in micrometers (use the formula for cell size determination as done in Unit 2 Microscopes). Put this information in Figure 4-4.
  3. Dispose of your slide in the sharps container on your bench.

Precautions

  • Make sure to use only a very small amount of bacteria.
  • Tap the bacteria onto a slide with the dowel, do NOT rub.
  • Dyes will stain your clothing. Make sure to have your lab coat buttoned up the front.

C.View Your Stained Bacteria:

Use your microscope to view your stained bacterial cells at a TM of 1000X. If you can see blue cells that are a distinct shape and arrangement, and that are distributed evenly across a field of view then you have had a successful simple stain with Methylene blue.

Figure 4-5. Results and drawing or photo for your simple stain, using methylene blue

Name of specimen: ______
TM used:______
Field diameter: ______
Number of cells that fit across: ______
Cell size in mm:______
Cell size in μm:______
Cell shape: ______
Cell arrangement:______/ Insert photo or draw the results of your simple stain here.

III.Day Two

Streak isolation Results.

Draw or insert a photograph of your streak isolation results here:

Do you have two different types of colonies?

If yes, briefly describe each colony type. Practice seeing the differences as you will need this skill for the unknown identification project in Unit 13.

Hold the plate up to the light. Can you see through the plate around any of the colony types?

Note which types have this clearing.

Post- Lab QuestionsName ______

  1. Describe three characteristics of microbes that can be detected using a simple stain like Methylene Blue.
  1. Explain in terms of charge attraction why Methylene Blue dye will stick to bacterial cells.
  1. What is the purpose of a streak isolation?
  1. What is a pure colony?
  1. How did your streak isolation from the mixed broth compare to your first streak isolation?
  1. Did you get isolated colonies? If not, what could you do differently next time?

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