9: Introduction to the Bacterial ID Project

Learning Outcomes

By the end of this laboratory (3 lab periods) you will be able to

• Explain the difference between selective and differential media
• Describe the differences between TSA, PEA, and MAC media
• Verify the existence of a pure culture by Gram staining.

Introduction

Today we will begin the capstone project of the microbiology laboratory - the Bacterial ID project. Today you will be given a single tube containing a mixture of one Gram-positive and one Gram-negative organism. Over the next six weeks, you will first isolate the two organisms, generating two pure cultures, and then perform extensive biochemical tests to determine which two organisms you have been given.

The identification of microorganisms in clinical samples used to be based solely on the kinds of testing we will be performing. However, with the advent of rapid genomic sequencing, clinical laboratories can often both identify organisms and determine their sensitivity to common antibiotics in a single step. Regardless, there is a lot to be learned about the biology of microorganisms in these biochemical tests and it remains a worthwhile exercise for us. Most microbiology students find this project to be a fun challenge!

Table 1: Possible Unknown Organisms

Possible Unknown Organisms
Gram-positives	Gram-negatives
Bacillus subtilis	E. coli
Staphylococcus epidermidis	Proteus mirabilis
Bacillus cereus	Providencia stuartii
Corynebacterium xerosis	Salmonella enterica
Bacillus megaterium	Proteus vulgaris
Lactococcus lactis	Shigella sonneii
Micrococcus luteus	Klebsiella pneumoniae
Enterococcus faecalis	Klebsiella aerogenes (formerly Enterobacter aerogenes)
Staphylococcus aureus	Citrobacter freundii
	Morganella morganii
	Alcaligenes faecalis

Overview of the isolation process

Your goal for the first 3 lab periods is to successfully isolate your gram-negative organism from your gram-positive organism. Hopefully, you’ve gained confidence over the past few weeks in performing high-quality streak plates! This skill will be extremely valuable during isolation. We will also employ the use of two types of “selective media” to stack the odds in favor of successful isolations. In today’s lab, you will be streaking out your unknown mixture on three types of media:

1. TSA (Trypticase Soy Agar)
2. PEA (Phenylethyl alcohol agar)
3. MAC (MacConkey agar)

You will also prepare a gram stain from your mixed-culture broth to get a first peek at your two organisms!

In the second lab period, you will be evaluating the growth on TSA, PEA, and MAC, and Gram-staining individual colonies on those plates. If you have successfully isolated one or both of your organisms, you will make a slant(s) of the pure culture(s) (Figure \(\PageIndex{1}\)).

There will be a third and final lab period to complete the isolation of both unknowns and confirm that you have one Gram-positive and one Gram-negative by Gram-staining in order to move on to biochemical testing.

Figure \(\PageIndex{1}\): In the course of this lab we will isolate your Gram-negative and Gram-positive organisms and subculture them on TSA slants.

TSA (Trypticase Soy Agar)

TSA is a general-purpose media that is non-selective. Both of your organisms (Gram-positive and Gram-negative) will grow well on TSA. We use TSA instead of Nutrient Agar (NA) because TSA is a slightly richer medium and our more fastidious unknown organisms grow more reliably on TSA than NA.

PEA (Phenylethyl alcohol agar)

Phenylethyl alcohol agar is a type of “selective” media. When media is selective, it allows some organisms to grow, while others are prevented. In the case of PEA, the alcohol is disruptive to the outer cell membrane of Gram-negative cells and they have a very difficult time growing under the conditions that are present in PEA. Thus, PEA selects for Gram-positive organisms and against Gram-negative bacteria.

PEA’s selection is not perfect. Some of our Gram-negative bacteria do grow a little bit on PEA, so it’s extremely important to perform your streak plate correctly in order to grow individual colonies that have originated from a single organism. You will want to confirm that these colonies are truly Gram-positive! Ideally, you will use only part of the colony to create your smear, and once you’ve confirmed that it’s correct, you can use the remainder to make your slant.

MAC (MacConkey agar)

MacConkey agar (MAC) is also selective, but in this case, it selects for Gram-negative bacteria and against Gram-positive organisms. In the case of MAC, bile salts and crystal violet dye prevent the growth of Gram-positives. MAC reliably selects against Gram-positive bacteria, but you will still want to confirm that these colonies are truly Gram-negative! Again, you will use only part of the colony to create your smear, and once you’ve confirmed that it’s correct, you can use the remainder to make your slant.

MAC is also “differential”. It differentiates between organisms that ferment lactose and those that do not ferment lactose. As we learned in the lecture, acids are commonly produced during fermentation, and these acids will lower the pH of the surrounding medium. MAC also contains a dye called “neutral red” which is a pH indicator. As the pH drops below pH 6.8, the dye turns a pink or red color resulting in distinctive pink or purple colored media. Organisms that do not ferment lactose leave the media looking brown (Figure \(\PageIndex{2}\)).

Figure \(\PageIndex{2}\): MacConkey media inoculated with a lactose fermentor (left) and a non-fermentor (right). Medimicro, Public domain, via Wikimedia Commons

Materials

• Mixed bacteria culture containing one Gram-positive and one Gram-negative bacteria
• Inoculating loop
• Bunsen burner
• Gloves
• 1 x TSA Plate
• 1 x PEA Plate
• 1 x MAC Plate
• Gram Staining Materials
• Microscope with oil immersion lens
• TSA slants (at least 2 per student)

Experiment

Day One:

1. Obtain and label plates (TSA, PEA, and MAC). TSA and PEA look alike, so be sure to label them with a Sharpie as you collect them (Figure \(\PageIndex{3}\)).
2. Obtain your unknown mixed culture (containing one Gram-positive and one Gram-negative bacteria) from your instructor.
3. Gently resuspend the mixture to evenly distribute the organisms.
4. Gram stain the mixed culture to see the initial morphology of bacteria when together. Be sure to carefully mix the broth to resuspend any bacteria that have settled to the bottom of the tube. Using a sterile Pasteur pipette, put three drops onto your microscope slide and dry on the hot plate to prepare your smear from the broth. Draw or photograph your initial Gram-stain to include in your final report.
5. Using aseptic streak plate technique, streak the mixed culture onto each of the three plates.
6. Incubate the plates at 37 degrees C for 48 hours, or at 30 degrees C for 5 days.

Figure \(\PageIndex{3}\): Uninoculated MAC, PEA, and TSA media. Note that you cannot tell TSA and PEA apart by looking at it! Be sure to bring your sharpie with you and label each plate as you collect it.

Day Two:

1. Remove plates from the incubator. Observe and record the colony morphology. What color are the colones? How big are they? Does either of your organisms exhibit swarming characteristics? Can you tell your two organisms apart by looking at their growth (often you can)?
2. Photograph EACH plate carefully - these pictures are a required element of your final ID project!
3. Using a sharpie, make a circle around colonies that are likely to be your Gram-positive and your Gram-negative organism. If the colonies are large enough, use only half (or less) of the colony to make bacterial smears for staining.
4. Gram-stain your smears, and confirm that you have colonies that contain only your Gram-positive bacteria and only your Gram-negative bacteria with no contaminating organisms. In addition to a uniform Gram-stain, observe the cell size and shape. Are you confident that the colony you selected contains only one organism?
5. Draw or photograph your Gram-stains. These pictures are a required element of your final ID project!
6. Once you have confirmed that you have isolated colonies of your Gram-positive bacteria and your Gram-negative bacteria, use the SAME colonies that you Gram-stained to create two slants - one that contains your Gram-negative bacteria and one with your Gram-positive bacteria.
7. Incubate the slants at 37 degrees C for 48 hours, or at 30 degrees C for 5 days.
8. If cultures are not pure, re-streak a colony onto the appropriate isolation plate - you have one additional lab period to successfully isolate any organisms that have eluded you so far. Incubate any new plates at 37 degrees C for 48 hours, or at 30 degrees C for 5 days.
9. Save all of your plates - your instructor will provide the class with a bin to store them in for the duration of the project. You may need them again if at some point your slants become contaminated.

Day Three

You are not finished with your isolations until you have confirmed that one of your slants contains your purified Gram-negative organism and the other contains your purified Gram-positive organism. Be sure to Gram-stain both slants before proceeding to the next lab. Note: It is very easy to accidentally contaminate your slants by using poor aseptic technique when making Gram stains or subculturing. Be fastidious with your technique EVERY time you stick a loop into the slant for any reason!

Data

By the end of this lab, you should have

1. Your Gram-positive and Gram-negative organisms on separate slants.
2. Photographs or drawings of your initial Gram-stain of the mixture
3. Photographs of all three of your streak plates (TSA, PEA, and MAC)
4. Photographs or drawings of your Gram-stains of your isolated colonies
5. Photographs or drawings of the Gram-stains of your two slants.

All of this data will be part of the final report you submit for the Bacterial ID project.

Creating a Test Table

This is a great time to begin working on your final report! You do this by creating a “test table” where you can begin collecting and interpreting all of your data. You will be turning in this table twice before submitting your final project. There is a Bacterial ID Project Template that you can copy and use if you find it helpful. You can also create your own table using any word-processing program.

For now, you can record all of your results so far. This table includes some examples to get you started, but you will want to put everything in your own words and add both pictures and written descriptions to the table.

Bacterial ID Project Test Table - Example

Table 2: Example Data Table for Unknown Project Report

Name of TEST	Name of media, description - how it works What does a + result look like? What does a - result look like?	Gram - Test Result, description, and pictures	Gram + Test Result, description, and pictures
Growth on TSA	An all-purpose medium that is non-selective. Both Gram-positive and Gram-negative organisms should grow.	Put the picture of your TSA plate here! Include a written description!	Put the picture of your TSA plate here! Include a written description - morphology, color, etc!
Growth on PEA	fill in with your description!	No growth.	Put the picture of your PEA plate here! Include a written description - morphology, color, etc!
Growth on MacConkey Agar	Bile salts and crystal violet inhibit the growth of gram-positive organisms, organisms that ferment lactose reduce the pH and turn the neutral red dye pink. An organism that is both gram - and ferments lactose grows and turns pink. An organism that is both Gram- and does not ferment lactose grows and does not change color. An organism that is gram-positive does not grow.	Put the picture of your MAC plate here! Include a written description - morphology, color, etc!	No growth
Gram Stains	describe how these work in your own words and what you expect to see from a G+ and G- organism	Gram stain picture here! Describe the cell morphology too!	Gram stain picture here! Describe the cell morphology too!