BIOL240 Chapter Notes - Chapter 1: Gram Staining, Crystal Violet, Gram-Positive Bacteria

88 views11 pages

sapphirecamel452

4 Aug 2015

School

University of Waterloo

Department

Biology

Course

BIOL240

Professor

Cheryl Duxbury

For unlimited access to Textbook Notes, a Class+ subscription is required.

Document Summary

The purpose of this lab was to understand and use the aforementioned staining techniques to identify different types of bacteria among the four used. Performing these experiments helped to solidify and understanding of when and in what situations a scientist would use the different techniques. When investigating microbes in a lab, one of the most important steps is to perform staining techniques on the cells in order for them to be easily observed even under a microscope. Without staining, bacteria may not contrast against the slide medium enough to be seen (prescott, 2001). The use of a stain allows scientists to easily identify the shape, size, and grouping of microorganisms. Stains are simply dyes that are able to highlight certain structures due to their chemical composition. There are a variety of different types of stains that are commonly used for specific situations.

Related Questions

2.Conclusion:

What have you learned from your study?

Interpret your data/ results:

Do your results support of disprove your hypothesis?

Compare and contrast your result with those of previous research.

Significance of the results: âBig Pictureâ

What is the global impact of your results?

What questions remain unanswered?

What kinds of experiments can come out of your study?

i have lab for biology and i need help with the Lab Report

the lab name is Bacterial Transformation Lab

1. Introduction:

Introduce the background and the nature of the problem your experiment is investigating.

Summarize the current knowledge of your subject.

Use examples of other research that have examined the same or a similar topic as your experiments.

Ending your introduction:

State your hypothesis

State your predictions

Give a brief statement about how you tested your hypothesis.

please help me with it is important for my lab class tomorrow

that is the lab i put the info please help me for the intro and for number 2 please be clear please

i would be happy if you help me it is due tommorrow i realy need your help on the intro and number 2 please be clear and show clear work hope you do it because it is due tomorrow

Bacterial Transformation Lab

Genetic engineering is one of the newest applications of basic knowledge in biology. It involves the isolation of specific genes from one organism and the insertion of those genes into a second organism of the same, or even different, species. Remember that a gene is a piece of DNA which codes for a product, usually a protein, which gives an organism a particular trait. The development of these techniques has generated considerable excitement and answered many questions, but it has also raised questions. Many scientists and others see genetic engineering as offering opportunities to make life better, for example, by exploring how genes are regulated, by curing diseases of genetic origin, by adding desirable traits to agricultural crops such as the ability to fix nitrogen in corn and pest resistance in cotton, and by genetically altering bacteria with genes enabling them to digest oil spills.

For almost 100 years, it has been possible to isolate DNA from cells, but only in the last 40 years or so has its function been understood. Your textbook discusses the work of such investigators as Fred Griffith and Oswald Avery, who demonstrated that if DNA was isolated from a pneumonia-causing strain of bacteria and added to cultures of nonvirulent bacteria, the recipient cells were transformed into virulent types that caused pneumonia. These transformation experiments were the first to show that genes could be artificially transferred in the laboratory. Since the time of those experiments (Avery's work was done in the 1940s), our knowledge of gene structure, function and control has increased astronomically. The application of this knowledge has created the field of genetic engineering.

Modern genetic engineering techniques depend on three critical factors:

A suitable host organism in which to insert the foreign gene;

A vector to carry foreign genes into the host; and

A means of isolating the host cells that have taken up the foreign gene.

The host organism

The most commonly used host organism is the gut bacterium Escherichia coli (E. coli). Its chromosome is a single large circular DNA molecule, called the genomic DNA, containing about 4 x 10⁶ base pairs. Not every cell will take up foreign DNA when exposed to it, but E. coli grows rapidly, dividing in less than 20 minutes, so that a single cell can give rise to a billion genetically identical descendants in a matter of 10 hours. Thus, a few transformed cells can provide a large number of offspring within a day. The strain we are using is K-12.

The vector

A vector is a DNA molecule used as a vehicle to carry genes from one organism to another. This DNA molecule can be modified by gene-splicing techniques using restriction enzymes and ligases so that it carries the genes of interest (essentially a cut-and-paste operation). Sometimes viruses are used as vectors, but for E. coli, plasmids are often used. A plasmid is a small circular DNA molecule containing 1000 to 200,000 base pairs. Plasmids exist naturally in many strains of E. coli and other bacteria. Plasmids replicate as the cell grows, independent of replication of the genomic DNA. These plasmids are transmitted to each of the product cells during cell division. Plasmids carrying genetic information that is beneficial to the host cell are maintained in a given population by natural selection. Plasmids frequently carry genes that confer antibiotic resistance, an obvious benefit.

When E. coli cells are exposed to plasmids or any other foreign DNA, some of the cells, called competent cells, will take up the DNA. We can induce cells to become competent by treating them with divalent cations, such as Ca⁺⁺, followed by a brief exposure to high temperature, called a heat shock treatment. Exactly how this treatment causes cells to become competent is not understood, but it is described as making the membranes "leaky" so that large DNA molecules (like plasmids), which would not normally pass through the cell membrane, can enter the cell. This treatment does not make all cells competent, but it greatly increases the number of competent cells. Once in a cell, the plasmids replicate and are passed to the product cells during cell division.

The selective medium

The last requirement for genetic engineering is a means of isolating host cells that have taken up the gene of interest. The techniques used depend on the growth requirements of the host organism. For example, each species of bacteria has particular nutrient requirements. The composition of the growth medium (agar or broth) can be modified to allow growth of only certain bacteria -- for example, by adding or removing nutrients or other chemicals from the medium mixture. This modified medium is called a selective medium, because it selects for the growth of only some species. By introducing an antibiotic such as ampicillin into the medium in which the bacteria are grown, you will select for growth of only those bacteria carrying the antibiotic-resistance gene on newly acquired plasmids. In a matter of days, billions of antibiotic-resistant cells, all with the plasmid, can be grown.

Genetic engineers use a modification of this technique to insert a foreign gene into E. coli. In simplified terms, the plasmid (already carrying an ampicillin resistance gene) is modified by inserting the gene of interest. The gene of interest has been isolated from another source; for example, the gene for human insulin has been isolated from the human genome. The modified plasmid, carrying the inserted gene, is then mixed with competent E. coli and the bacteria are placed in a medium containing ampicillin. Only those cells that have the plasmid will grow in the medium, and they are also the cells carrying the insulin gene (in their plasmids). These selected cells can now be used to manufacture insulin, which can be used for insulin therapy by diabetics.

The genes

In the following experiment, you will insert a plasmid into E. coli. The plasmid you will use carries the gene bla, which codes for a protein (beta-lactamase) that is secreted by the bacteria, inactivating the ampicillin in the agar and allowing for bacterial growth. The plasmid also carries an indicator gene. The indicator gene we are using was isolated from a bioluminescent jellyfish, Aequorea victoria. It codes for Green Fluorescent Protein (GFP).

The GFP gene has been modified to include a regulator system that can be used to control the expression of the gene. Gene expression in all organisms is carefully regulated to allow for adaptation to differing conditions and to prevent wasteful overproduction of unneeded proteins. The genes involved in the breakdown of different food sources are good examples of highly regulated genes. For example, the sugar arabinose is both an energy source and a carbon source for bacteria. The bacterial genes that make digestive enzymes to break down arabinose for food are not expressed when arabinose is not in the environment. But, when arabinose is present, these genes are turned on. When the arabinose runs out, the genes are turned off.

Arabinose initiates transcription of these genes by promoting the binding of RNA polymerase. In the genetically engineered pGLO DNA, some of the genes involved in the breakdown of arabinose have been replaced by the jellyfish gene GFP. When bacteria with this plasmid are grown in the presence of arabinose, the GFP gene is turned on and the bacteria glow green in UV light. When arabinose is absent form the growth medium, the GFP gene remains turned off and the colonies appear white. This is an example of the central molecular framework of biology in action: DNA ? RNA ? protein ? trait.

In summary, you will render E. coli cells competent to take up plasmid DNA. The plasmid carries two identifiable phenotypic markers:

The beta-lactamase or bla gene codes for resistance to the antibiotic ampicillin. We will use this to select for bacteria that have taken up the plasmid.

The pGLO gene codes for Green Fluorescent Protein (GFP), which is switched âONâ if arabinose is present.

Safety Procedure

For your own safety and the safety of others, it is essential that you follow all instructions about handling bacteria and disposal of materials. When you finish work for the day, swab your table with disinfectant. WASH YOUR HANDS thoroughly with soap before leaving the lab for any reason, and at the end of the day after you have cleaned your table. Report any spills to your instructor.

List of Materials

Prepared agar plates (2 LB, 2 LB/amp, 1 LB/amp/ara)

Transformation solution (50 mM CaCl₂, pH 7.4)

LB broth

1 pack of inoculation loops

Sterile transfer pipets

1 foam microtube holder w/ tubes

E. coli starter plate

1 red biohazard bag for loop disposal (common use â Loops/pipets only - NO PAPER!!!)

beigefox320

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

goldturtle749

BIOL240 Chapter Notes - Chapter 1: Gram Staining, Crystal Violet, Gram-Positive Bacteria

Document Summary

Get access

Related Documents

BIOL240 Study Guide - Final Guide: Gram Staining, Methylene Blue, Acid Dye

BIOL240 Study Guide - Gram Staining, Methylene Blue, Crystal Violet

BIOL240 Study Guide - Quiz Guide: Mycobacterium Tuberculosis, Gram Staining, Methylene Blue

Related Questions