BIO SCI M122 Lecture 35: Gram Staining
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A 47 year old female patient presents herself to you with symptoms of pneumonia. All indications are that the patient is suffering from an infection of Streptococcus pneumoniae, a Gram-positive bacterium. Since the patient is not allergic to penicillin, you prescribe a dose of amoxicillin, a semi-synthetic penicillin. This drug works by blocking the peptide cross-links, between the muramic acid subunits, of peptidoglycan chains. This leads to a destabilization of the bacterial cell, leading to its rupture.
However, after the patient finished her course of amoxicillin, no improvement in symptoms were reported. Perplexed, you decide to culture and examine the bacteria infecting her lungs. The test you order is a Gram stain test, which comes back as Gram-negative. You feel like a failure as a health care provider due to your misdiagnosis of this bacterium.
The same day, you put the patient on an aminoglycoside class antibiotic. This antibiotic is Gram-negative specific and works by shutting down ribosomes. You are dismayed and confused when this treatment also fails.
To get a full read on the disease, you have a barcoding DNA test done on the causative bacterium. The test comes back and it clearly shows that the bacterium is within the group of Gram-positive bacteria. With the results of this test, you come up with the theory that the patient is infected with a strain of Drug Resistant Streptococcus pneumoniae (DRSP), resistant to amoxicillin. You decide to treat the DRSP by administering intravenous vancomycin, which is indicated for the treatment of serious, life-threatening infections by Gram-positive bacteria. This treatment also fails.
In the end, you figure out that you were wrong about everything, and that there was a simple solution to the treatment of the patient.
Now knowing that the patient was infected with Mycoplasma pneumoniae, what is the likely explanation for the false Gram-negative test?
Before you answer this question you may want to know how Gram staining works. Briefly, the Gram stain is a differential stain meaning that you are using two dyes that stain different structures. Based on the structural differences of the bacteria, the dyes will interact differently with them, producing different results (colors). The Gram stain (Crystal Violet together with Gram's iodine) stains thick layers of peptidoglycan purple and thin layers of peptidoglycan very, very lightly purple. The second stain (or counterstain) is safranin. Safranin is a dye which interacts with and binds to lipid bilayers. Yes, safranin also stains the cytoplasmic membrane of Gram-positive bacteria, but the deep purple color of the Gram stain makes it impossible to see.
| A. | The Gram stain was able to penetrate the cell making it turn pink | |
| B. | The counterstain stained the cytoplasmic membrane, making it look like a Gram-negative bacterium | |
| C. | The counterstain stained the mycolic acid in the cellular envelope, thus making it look like a Gram-negative bacterium | |
| D. | The Gram stain lightly stained the present peptidoglycan (making it look pink rather than red). |
BIOTECHNOLOGY
Introduction
In this Experiment we will learn how to identify the unknownbacterium according to the case study given as you start to performyour procedures and following the steps. Also, we will be learningthe outcome of a specific case scenario of immunity. We will beperforming different examples of media and observing the reactionafter inoculation and adding different reagents as it produce somechanging of color diagnose to a negative or positive results. Also,we will conduct different types of biochemical testing includingIndole production, Citrate Utilization, Methyl red test, andMannitol Fermentation.
In this case scenario we are trying to figure out theinfestation of food at the warehouse causing a death of theplaintiff mother. The plaintiff is accusing the warehouse forselling these infested products by insects. At the warehouseinspection is been done by swabbing and acquiring the type oforganism to be identified as evidence for the court finaldecision.
One of the major findings in this experiment is that itwas negative gram stains and the Indole and Methyl Red Test had apositive reaction. Additional testing was perform which show theresult of only negative on Lactose Fermentation and the remainingthree additional testing add up to a positive. The outcome of thisinvestigation scenario could be a negative result to the casescenario presented about infested products by insects since nounknown bacteria was left to bediagnosed.
Procedure
The following steps are requiring completing this labexperimental. First make sure you open the Virtual UnknownSoftware. Complete conducting using Indole production and CitrateUtilization, Methyl Red test choosing from your media option. Alsomake sure you open the reference book for each procedure to beinformed of the lab steps and decision making such as positive ornegative and its color after inoculation.
Observations and Results
Result + or - | Appearance after Incubation | |
Gram Stain | - gram rods | N/A |
Indole Production | + positive | Yellowish-after incubation & adding Kovacâs reagent changingto a cherry red color indication of positive |
Mannitol Fermentation | - neg | Red |
Methyl Red Test | + pos | Turbid yellowish after incubation and adding methyl red reagentchange to red positive. + |
Citrate Utilization | - neg | Greenish after incubation. No reagent added. |
Additional tests performed are listed below:
Result + or - | Appearance after Incubation | |
Lactose Fermentation | - neg | Red- no reagent added |
Sucrose Fermentation | + pos | Yellowish- no reagent added |
| Phenol Red Rhamnose (Lactose Steps) | + pos | Yellow- no reagent |
Phenol Red Xylose (Sucrose Steps) | + pos | Yellow- no reagent |
Bacterium identified as: You may want to do some additionaltests to conclusively identify your bacterium. Some good ones to domight be lactose and sucrose fermentation, like you did last week.Remember, they use Phenol Red Lactose Broth and Phenol RedSucrose Broth and yellow= positiveand red =negative. After you do those two tests,you may want to do Phenol Red Rhamnose and Phenol RedXylose, which are set up the same way as the sucrose andlactose tests, they just have different sugars. Be sure to recordyour test results!
Discussion
Identify the bacterium â Make sure you look in the VirtualUnknown lab report to make sure you got the right organism. Afteryou have made your identification, look at the lab report. You cando this by going to the View menu in Virtual Unknown and selectingLab Report. You can double check with me that you are researchingthe right one.
Once all observation was performed including the additionaltesting I was left without an Unknown organism to be identified.This only leave me with a diagnose to state that no evidence wascollected to accused the warehouse as being infested with insectsaccording to the case study.
Explain what features of this organism make it an idealcandidate host for your insecticide. (Hint: remember you will needto infect insects with the bacteria to deliver the insecticide.)âFind out where the unknown organism was isolated from.
Describe in detail the steps you will take to insert the targetgene into the bacterium. - Figure 8.1 of your book should help youwith this.
Conclusion
< Within a few sentences, provide a concluding statementabout the results of your laboratory>
Virtual Lab Report
Student:
Instructor: Test User
Section:
Unknown: BiotechUnknown
Identification Information
Assigned Unknown: Identification not yet complete
Identified Unknown: TBD
Gram Reaction & Morphology: Gram Negative Rods
Auto-Inoculation Used: Yes
Test Detail (Chronological Order):
1 Gram Reaction {-} Eliminated {53}Remaining {71} Recorded 1 time(s)
Observations: none
2 Indole production {+} Eliminated {44}Remaining {27} Recorded 1 time(s)
Observations: none
3 Mannitol fermentation {-} Eliminated {19}Remaining {8} Recorded 1 time(s)
Observations: none
4 Methyl red {+} Eliminated {0} Remaining{8} Recorded 1 time(s)
Observations: none
5 Citrate Utilization (Simmons) {-}Eliminated {2} Remaining {6} Recorded 1 time(s)
Observations: none
6 Lactose fermentation {-} Eliminated {0}Remaining {6} Recorded 1 time(s)
Observations: none
7 Sucrose fermentation {+} Eliminated {4}Remaining {2} Recorded 1 time(s)
Observations: none
8 Rhamnose fermentation {+} Eliminated {2}Remaining {0} Recorded 1 time(s)
Observations: none
9 Xylose fermentation {+} Eliminated {0}Remaining {0} Recorded
