BACTERIAL CULTURE TECHNIQUES LAB

Escherichia coli, a gram negative rod shaped bacteria that normally inhabit the human gut is extensively used in molecular biology laboratories. Particular E. coli strains used in the labs have been grown in invitro culture for many generations and as a result have lost their ability to colonize the intestines effectively.

E. colihas widely been transformed because of its simple prokaryotic structure, short generation time (about 20 minutes), and simple growth requirements.

Aseptic Technique

Aseptic technique refers to the procedure that minimizes the chance of introducing microorganisms from the environment to the bacterial culture. In order to minimize contamination of cultures with foreign bacteria and fungi, all glassware, culture media, pipettes, pipette tips andinoculation loops that come into contact with the culture are sterilized before using. Inoculation of cultures is performed under aseptic conditions and cultures are kept closed after inoculation.

The growth MEDium

The growth medium provides bacteria the nutrients needed for their growth as well as a means of handling bacteria for us. Media can either be solid or liquid broth. Solid media contain agar, a complex polysaccharide derived from red algae. E. coli has simple nutritional needs and is commonly grown in LB (Luria-Bertani) media.

The growth medium is sterilized by autoclaving at 121 C/15 psi for about 20 mins. Autoclaving destroys all organisms (including bacterial endospores), but not prions.

  1. Preparation of LB agar plates (plates have been prepared for you)

LB agar – 1 Liter

10gTryptone

5gYeast extract

10gNaCl (mw 58.44)

  • Add ~ 700 ml of deionized water to a beaker. Add the above ingredients and stir on a magnetic plate with a stirring rod until all solids are completely dissolved. Adjust pH to 7.4 with NaOH. Transfer to a graduated cylinder and make final volume 1 liter.
  • Mix well and aliquot 100 ml into ten bottles. Add 1.5 g agar to each bottle containing 100 ml LB. Keep caps loose and autoclave 20 min at 121 C/15 psi.
  • Pour plates from one or two bottles immediately after removing from the autoclave while the media is still liquid. Alternatively, if allowed to cool and solidify, LB agar can be melted in a microwave in pulses.
  • To pour plates, disinfect bench top with isopropyl alcohol. Spread the plates on the bench top with lids in place. Open one plate at a time and pour autoclaved LB agar until the bottom of the plate is covered. You may swirl the plate to get it covered. Let LB agar solidify for about 15 mins. Dry plates in a 37 C oven for 30-45 mins with lids partially opened.
  • Label plates on the bottom. Store in a plastic bag in the refrigerator.Store plates upside down to prevent condensation from falling back on the media.

Streak plate method for isolation of individual colonies

When using E. coli cultures, it is important to work with a pure culture, or a culture consisting of a single strain that is genetically identical. To ensure a pure culture, cultures are started from a single colony of bacteria. An individual bacterial colony represents a population of cells derived from a single mother cell divided many times by binary fission, and hence is “pure”. In this lab, streak plate method is used to isolate individual bacterial colonies.

Exercise:

Safety concerns and precautions:

  1. You will use an incinerator heated for about 10 minutes. Incinerator will be hot. Do not leave inoculation loops resting in the incinerator. The handle will burn quickly.
  2. Make sure agar plates are handled and stored with the agar side up to prevent condensation from forming on the agar surface.
  3. Do not leave plates open to the air.

Materials needed (per person):

2 LB agar plates

Wild typeE. coli culture

Inoculation loop

Incinerator (heated for about 10 mins)

Method:

  1. Each person will prepare two streak plates. Label the bottom of agar plates (agar side) with your last name with first initial, date and description of type of culture. Divide the plates into three quadrants. Place plates on the desk with the agar side up.
  1. Sterilize the inoculation loop by holding the wire portion of the loop in the incinerator for 5-10 seconds until it glows a bright orange. Remove the inoculation loop from the incinerator and allow to cool a few seconds while holding with the handle.
  1. Place the E. coli culture plate on the desk, agar side up. Lift the side containing agar and lightly scrape a clump of cells from a colony. Do not dig into the agar plate. Return the agar side of the culture plate to the lid.
  1. Lift the agar side of your LB plate and streak quadrant one with zig-zag motion as demonstrated by the instructor. Be sure to glide your inoculation loop on agar and not gouge the agar. Sterilize the inoculation loop.
  1. Drag bacteria from the first quadrant into the second and spread the bacteria in the second quadrant with zig-zag motion. Sterilize the inoculation loop.
  1. Drag bacteria from the second quadrant into the third quadrant and spread the bacteria in the third quadrant with zig-zag motion. Sterilize the inoculation loop. Return the agar side to the lid.
  1. Stack the four plates (agar side up) from you and your partner first and tape them together on one side. Then stack the plates from the whole class and incubate at 37 C in the incubator.

Day two:

  1. Observe your LB streak plate. Do you have well isolated colonies on the plate? Typically the first quadrant will have cell masses that run together and form an even lawn of growth. Individual colonies start to appear in the second or the third quadrant. Colonies are distinct, uniform and circular formed by a single mother cell.

Gram Staining Technique

The Gram stain procedure, developed by Hans Christian Gram in 1884, is a differential staining technique. It allows the differentiation of bacteria into one of the two groups based upon the color of the bacterial cells at the end of the procedure. The color of the cells at the end of the staining procedure depends on the differences in the structure of the cell wall.

Those that stain purple blue at the end of the procedure are said to be gram-positive. These bacteria have several layers of peptidoglycan in their cell walls.

Bacteria that stain pink after the gram stain are said to be gram-negative. These bacteria have an outer lipid membrane and a small amount of peptidoglycan in their cell walls.

Materials needed (per pair):

Gram stain reagents

Escherichia coli culture

Staphylococus epidermidis culture

Inoculation loop

Incinerator (heated for about 10 mins)

Method:

Each pair will prepare three smears. One smear will contain E. coli, one smear will contain S. epidermidis, and the one smear will contain a mixture of the two species. These three slides will be stained using the Gram stain procesure.

Smear preparation:

  1. Label the slides with your names and the name of the organism.
  1. Place a loopful of water on each slide
  1. Sterilize the inoculation loop. Hold the wire portion of the inoculating loop in the incinerator for 5-10 seconds or until it glows a bright orange. Remove the inoculation loop and allow it to cool a few seconds.
  1. Transfer a sample of bacteria from the Petri dish to the respective slide. Lift the side containing the agar out of the lid. Distinct colonies should be visible on the surface. Lightly scrape a sample of a colony from the agar. Try not to dig into the agar. Close the plate by returning the plate to the lid. The plate should remain closed with the agar side up.
  1. Transfer the bacteria from the loop to the drop of water on the slide and mix. Spread the sample over an area about the size of a quarter. The thinner and more spread out it is, the faster it dries and better it stains.
  1. Sterlize the loop.Repeat steps 2-5 for the other bacteria. Repeat steps 2-5 for the mixture of bacteria. Be sure to sterilize the loop between transfers of the two species. Add both bacteria to the drop of water before mixing.
  1. Allow all slides to air dry.
  1. Heat fix the slide by placing the back of the slide close to the incinerator opening for 5-10 seconds. Be careful as the incinerator will be hot!. Heat-fixing kills the bacteria and cause them to fix to the slide.
  1. Proceed to the staining technique.

The Gram Stain Procedure:

  1. Flood each stain with crystal violet for 1 min, then rinse with water. Shake off excess water into the sink. There is no need to blot the slide.
  1. Flood each stain with Gram’s iodine for 1 min, then rinse with water. Shake off excess water into the sink. There is no need to blot the slide.
  1. Flood each smear with 95% ethanol (decolorizer) for 3-5 seconds. Quickly rinse with water, shake excess water off. There is no need to blot the slide.
  1. Flood each stain with Safranin for 1 min, then rinse with water. Shake off excess water into the sink. Blot the slides with bibulous paper.
  1. Observe slides under the microscope.
  1. Determine Gram morphology. Draw your results.