Streak Plate And Viable Cell Count Biology Essay

Aim and debut should expose penetration into what the run home base and feasible cell count method are employed to accomplish. They should besides present MacConkey agar and how its selective and differential belongingss allow the features of the trial being to be determined.

Escherichia coli ( E.coli )

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The purpose of this experiment is to let a certain bacteria to split and multiply enabling us to see the bacterium in a individual cell construction. E.coli is one bacteria that is good for such an experiment. E.coli can be said to be both bad and harmless, some E.coli bacteriums have are extremely toxic and can harm worlds and animate beings. However, the bulk of E.coli strains are comparatively harmless with low toxicity. These harmless strains of E.coli are found of course happening in the human organic structure, particularly in countries such as the human bowels. Some E.coli can even profit their hosts ; they do this by bring forthing specific vitamins. It is for grounds like the 1s mentioned why E.coli is an appropriate bacteriums to utilize for this experiment. Another ground is that E.coli bacterial cells have an mean bacterial size of 2um ; this can be seen under a light microscope. Other bacteriums nevertheless may be even smaller and may necessitate a larger microscope for sing or even an negatron microscope. Besides the incubation period for E.coli to multiply and turn quickly is n’t really long and temperatures are n’t excessively high or excessively low. E.coli can be incubated nightlong at 37oC and so stored at 4oC until its demand.

The technique usage to pull strings and insulate the E. coli bacterium is known as the run home base process. The technique was developed to let bacteriums to multiply and bring forth many settlements, during the incubation period, depending on the sum of bacteriums present. Each settlement will incorporate 1000000s of bacteriums cells derived from a individual parent cell. ( Talk in more item about this process ) . We will be utilizing this technique to let the E. coli to multiply and split dividing itself into settlements.

The feasible cell count, besides known as viability count, is a method used to find the figure of life cells within a suspension, in this instance E. coli. To obtain an apprehension of how much E. coli cells are present in a sample this method must be put into action. ( Expand )

The MacConkey agar is specifically designed to let gram negative bacteriums to turn ; it ‘s a formula of many substances such as gall salts, Na chloride proteose and many more. One of the belongingss of the MacConkey agar is its selective isolation and designation of bacteriums ; it is a medium that allows us to separate Gram-negative bacteriums. E. coli is a rod shaped gram negative bacteriums, so utilizing the MacConkey agar home base to multiply it would be appropriate, the agar will besides do the E. coli to alter coloring material from pink to red, and this is an indicant of gram negative bacteriums present.

A Nutrient agar is a growing medium used for the cultivation of bacteriums, this specific agar remains solid even at high temperatures. The gram-stain technique was developed for sing cells clearly under a microscope and to enable us to set up their constructions. It is a really simple process of merely adding 4 different substances consequently, nevertheless one of these substances is toxic to worlds therefore the process must be carried out in a fume goon. The gm discoloration method was introduced by

It is of import for scientists and trefoils to cognize the construction and map and individuality of bacteriums and viruses, it is for grounds like this why such experiments are carried out. Without such processs so many bacteriums and viruses would n’t be known and could distribute and go out of control.

Methods:

Explain why each process was done – high spot key points – province any divergence from protocol – papers any mistakes or troubles you had with the technique

Discuss the importance of sterile technique and what stairss could be taken to forestall taint during use of bacteriums

All methods were making under sterile conditions ; the ground for this is to forestall taint of the bacteriums during its use. Many mistakes could originate if sterile conditions are n’t used, finally ensuing is wrote consequences.

Streaking bacteriums on MacConkey agar method:

Prior to the experiment, an E. coli sample was made and given to during the practical.

Hazard appraisal:

Materials used:

10ml liquid civilization of E. coli – the bacteriums sample to be usage in this practical

Sterile plastic cringles – used for reassigning E.coli bacterium from one topographic point to another uncontaminated.

MacConkey agar home base – used to let E.coli bacteriums to turn as it provides energy resorts and support

Sharps bin for loops etc. – these are used to maintain the laboratory country every bit uncontaminated as possible and to do certain bacterium does n’t distribute

Marker pens and labels to label the home base

Measure 1: utilizing a unfertile plastic cringle I touched a given sample of E. coli and streaked an inoculant onto a MacConkey agar home base in a specific form ( see.. ) . This fictile cringle is so disposed of into the sharps bin.

Measure 2: utilizing another unfertile plastic cringle, I created a tally of parallel lines from the border of the initial runs

Measure 3: measure 2 was repeated 2 more times with a new unfertile cringle used.

Measure 4: a concluding run was made, making a simple run from the old runs into the Centre of the home base. The image below illustrates this.

The MacConkey home base was so given to the technicians to incubate.

It was of import to dispose- on the plastic expressions by puting them into the sharps bin because they are contaminated and if they touch any other surface it can take to the spread of bacteriums ensuing in major taint. Throughout this process plastic baseball mitts and a lab-coat were worn, besides to forestall taint.

Feasible cell counts:

Hazard appraisal

Materials used:

P1000 and P100 pipettes and tips – used to reassign certain sums of PBS etc.

Three Nutrient agar home bases

10ml unfertile PBS – buffer, used to keep the pH

Sterile plastic spreaders – to distribute the E.coli on the Nutrient agar home bases

Eight unfertile bijoux bottles – for dilutions

To get down the dilution, utilizing a pipette I transferred 900 ul of dilutants ( PBS ) in eight different unfertile bijoux bottles. The PBS ( phosphate buffered solution ) solution is a normally used buffer to keep a pH ; it is used in this practical because of its ability to help biological research. After the PBS was placed into the labeled bottles, utilizing a new unfertile tip for the pipette I transferred 0.1ml of E.coli liquid civilization sample ( orderly ) into the first bottle ( 10-1 ) . For the dilution to go on a new pipette tip was placed and 0.1ml of the 10-1 diluted E.coli was transferred to the 10-2 bottle, this procedure continued up till bottle 10-8. By making so the E.coli will go more and more dilute within the different solutions, because less E.coli is being added each clip. 10-5, 10-6 and 10-7 samples were so spread onto three different Nutrient agar home bases utilizing different unfertile plastic spreaders so taint would n’t happen. This was done by puting 0.1ml of each dilution onto the Centre of the agar home base and so distributing it over with a unfertile spreader. The agar home bases were labelled and given to the technician for incubation.

Gram Stain of bacterium from an stray settlement

Hazard appraisal

In order to stain the bacterium I selected an appropriate settlement to stain, the settlement must look to be uncontaminated and its visual aspect must evidently look grown.. After this process is complete, the bacterial cells will be seeable under a microscope.

Materials needed:

Bunsen burner – used to heat-fix bacteriums onto microscope slide

Saline ( PBS ) – emulsifier

Light microscope – to see bacterial cells

Lens tissue – to clean the lens

Immersion oil for light microscope lens – to let better position at 100x magnification

Stains – for Gram discoloration method

Before the bacteriums can be “ modified ” to be viewed under the microscope clearly, the microscope glass slide must be cleaned to forestall taint. After making so a bead of unfertile saline was placed onto the Centre of the slide, the saline bead was placed because it can emulsify any bacterial settlement that will be placed on top. To travel some of the bacteriums off the agar home base onto the slide, a unfertile cringle was used – I touched the bacterial settlement on the agar home base with the top of the cringle and so spread the bacterium into the saline bead doing it thin. Due to the wet of the liquid I let the slide dry so used a Bunsen-burner to heat-fix the bacteriums onto the slide by go throughing it through a few times so leting it to chill. Heat-fixing was done so that during the staining the bacterium or would n’t travel or fall off. Once that was complete the slide was moved to a research lab smoke goon where the staining can take topographic point, the follow 4 phase method was used: at foremost the bacterium sample on the slide was soaked in crystal violet for 30 seconds, after so it was rinsed with distilled H2O and drained. The 2nd portion is to soak the bacteriums with gram I ( mordant ) for another 30 seconds so rinse with distilled H2O and drain it. Gram I is a toxic substance ; it is for this peculiar ground why this portion of the practical was carried out in a research lab smoke goon. Acetone decolouriser was so added for 10 2nd and the bacterium was once more rinsed with distilled H2O and drained. The concluding portion is to add Safranin, a counter discoloration, to the bacterium. It was placed on the bacterium for 30seconds and so the bacterium was further rinsed with distilled H2O, drained, blotted and allowed to dry.

Substance

Duration

Further action

Crystal violet ( primary discoloration )

30 seconds

Rinse with H2O & A ; drain

Gram Iodine ( Mordant )

30 seconds

Rinse with H2O & A ; drain

Acetone/alcohol ( Decolouriser )

5-10 seconds

Rinse with H2O & A ; drain

Safranin ( Counter discoloration )

30 seconds

Rinse with H2O, drain, smudge & A ; dry

Stain was carried out in a research lab smoke goon due to the toxic gm I substance used. The crystalline plastic shield of the fume goon was lowered so that merely my custodies were inside covering with the chemical and biological substances. Baseball gloves were worn during this process so that no discoloration would come into contact with the tegument. When the slide was rinsed with H2O, it was rinsed gently with distilled H2O so that the bacteriums are non shifted.

After the staining was completed the sample can now be viewed under a light microscope and compared to other bacterial samples. The slide is placed on the phase with a bead of oil for submergence, the microscope is focused on 100x and the bacterium is viewed.

Consequences:

Should depict your findings in: prose/text, diagrams, tabular arraies and graphs which includes a description of growing features and how successful your sterile technique was

MacConkey agar home base consequences:

During the experiment there were no consequences to be noted as it was excessively early for anything to happen. After the agar home base incorporating the E.coli was incubated at 37oC and so stored at 4oC, its visual aspect was as expected. Colonies were separated, and as the runs moved on less E.coli was present. The settlements were good typical and were round in their form. The sample ab initio given was merely liquid, the consequence showed important growing of this E.coli liquid into 3D constructions. This indicates the growing of the bacteriums in a all right manner ; the 3D constructions appeared in a yellowing solid coloring material. Because the practical was conducted in sterile techniques no taint occurred. Aseptic techniques were successful in leting me to bring forth accurate consequences.

Feasible cell counts

My consequences:

The settlements that appeared on the alimentary home base had a badge coloring material ; visually they all appeared comparatively same sized and volume.

10-5

10-6

10-7

10-8

TMTC

46

1

For the 10-7 the computation for the figure of bacteriums in 1ml of the original civilization is:

( 1×107/0.1 ) ten ( X/1 ) [ cross multiply ]

0.1X = 1 ten ( 1×107 ) [ divide by 0.1 ]

Therefore X = 1.0×108

For the 10-6 the computation for the figure of bacteriums in 1ml of the original civilization is:

( 46×106/0.1 ) ten ( X/1 ) [ cross multiply ]

0.1X = 1 ten ( 46×106 ) [ divide by 0.1 ]

Therefore X = 4.6×108

The figure of bacteriums present in 1ml of the 10-5 civilization can non be calculated as there was no value noted ( TMTC ) .

X= figure of bacteriums. The figure of bacteriums present in 1ml of 10-6 dilution is 4.6×108 and in the 10-7 dilution civilization is 1.0×108.

Class consequences:

Pair figure

10-5

10-6

10-7

10-8

1

TMTC

46

1

2

95

9

0

3

TMTC

52

9

4

TMTC

23

1

5

34

2

6

84

11

4

7

TMTC

24

18

8

2

2

26

9

102

6

0

10

19

3

3

11

TMTC

140

15

12

TMTC

57

9

13

61

12

0

14

8

6

1

15

195

51

4

16

55

27

3

17

TMTC

94

11

18

TMTC

TMTC

TMTC

19

TMTC

TMTC

TMTC

20

TMTC

113

18

Entire

266

985

177

28

Average

66.5

57.9

9.8

7

Should depict your findings in: prose/text, diagrams, tabular arraies and graphs which includes a description of growing features and how successful your sterile technique was

To find the sum of bacteriums within a civilization a simple computation must be done utilizing my personal consequences for this experiment. There was no value for the 10-5 so this can non be done.

The consequence for 10-6 was 46, 46 ten 10 = 460ml. To gauge the sum of E.coli nowadays this is farther multiplied by 106, hence 460 ten 106 =

For the 10-7 consequence i? 7 x 10 = 10 i? 10 ten 107 =

However, I have selected some sensible consequences from the tabular array to cipher an norm.

Gram-stain of bacteriums from an stray settlement: ( position method figure 3 )

Gram stains assist us separate between microbic beings, for illustration gm negative bacteriums and gram positive bacterium. This method was developed to cognize the individuality of bacteriums present. ( See process for The Gram Stain in the methods subdivision ) .

During measure 5 of the Gram Strain Method above the undermentioned consequences were made when using the four different substances:

Substance

Coloring material after discoloration

Crystal violet ( primary discoloration )

Purple

Gram Iodine ( Mordant )

Purple

Acetone/alcohol ( Decolouriser )

Transparent ( dye was washed off )

Safranin ( Counter discoloration )

Reddish-pink

The visual aspect of the E.coli bacteriums under a microscope with 100x magnification was rather clear ; it had a rod-like construction with a reddish-pink coloring material. The rods were all more or less the same size, nevertheless some were packed together and others were on their ain.

Discussion:

Were the consequences the expected? Did the methods adopted achieve their purpose? How the experiments could be improved. Include background information, critical rating of consequences

Throughout all the experiments and procedures a lab-coat and baseball mitts were worn to avoid skin contact with bacteriums and harmful substances. Overall the purposes were accomplished and the consequences were as predicted.

MacConkey agar

The settlements were expected to be in such a signifier, bespeaking that it was E.coli present and that it has quickly multiplied into single settlements. This farther suggests that when E.coli is present under conditions where it could multiply, it multiplies by organizing a unit of ammunition settlement and spread outing from at that place. However, some of the settlements were stuck together doing it hard to number the figure of them present. What this means is that the growing of the bacterium was a success and the method adopted was accurate. The ground why some settlements were packed together may be the consequence of pressing excessively hard on the agar while streaking, with more streaking pattern more accurate consequences can be obtained with settlements being on their ain. The methods adopted for this practical achieved what was aimed for. After the incubation of the MacConkey agar plate the home base was stored for a hebdomad at a temperature of 4oC, this may hold changed the visual aspect in coloring material and in form of the formed settlements. Contamination of the agar home base may hold even occurred. An betterment to the experiment is to observe down consequences directly after incubation is finished.

Gram Stain consequences

After analyzing the microscope slide which contains the Gram Stained E.coli under the microscope its characteristics were obvious. There were many mean sized rods with a reddish-pink coloring material, some of these rods were packed together whist others were separated. Comparing this with another prepared sample of B.subtilis, the B.subtilis was a violet coloring material and has a longer and curving form, like all right togss. However some once more were packed together and others separated.

The violet coloring material of the B.subtilis indicates that it is gram positive, and the pink coloring material of E.coli indicates its gram negative. When the Gram Stain method was applied to the B.subtilis it evidently stayed violet though out, with E.coli it will bleach one time the decolouriser is added. The gm discoloration method is extremely effectual and efficient when covering with different bacteriums ; it helps place them to a great extent. B.subtilis remains violet throughout the Gram Stain process, this itself can be an indicant that it is a Gram positive bacteriums.

Bacterial cells have different types of cell walls, the gm negative and gram positive footings describe the nature of their structural differences. One of the of import differences is that Gram positive bacteriums have no outer membrane whereas Gram negative bacteriums do, the intent of this outer bed is to cover the peptidoglycan bed. When staining occurs the outer membrane of a gm positive bacterial cell wall becomes for good stained as the strain can easy perforate the thick peptidoglycan bed, so that if a decolouriser or distilled H2O is added the coloring material will stay violet. In the instance of the gm negative bacterial cell wall the discoloration gets attached to the far outer membrane bed ( lipopolysaccharide and protein ) , this bed decreases the incursion deepness of the strain on the peptidoglycan, so the discoloration can be decolourised or removed.

The diagrams below illustrate this.

Gram positive:

i? i? i?

Primary discoloration Mordant Decolourisation Counter-stain

Note: coloring material remains the same throughout add-on.

Gram negative:

Primary discoloration Mordant Decolourisation Counter-stain

Note: coloring material alterations i? i? i?

The purpose of the Gram Stain method was confirm that the bacteriums that was ab initio being dealt with was E.coli, after trials and consequences it confirmed that it was so the consequences were as expected and predicted.

The methods used for this process were successful at accomplishing good consequences, nevertheless some can be altered. For illustration, the E.coli used for this experiment was used from experiment figure one, non that that is a job but when the E.coli was incubated over dark and it had successfully multiplied it was stored at 4oC for rather a piece ( this experiment was carried out 1 hebdomad after the first 1 ) . This possible may hold altered the activity of the E.coli and besides its visual aspect. Many resources province that gm negative bacteriums should hold a pink coloring material after the counter-stain has been added and rinsed off. In this instance the E.coli bacterium in this experiment had rather a dark pink coloring material which was truly close to the coloring material ruddy, this visual aspect of coloring material was ocular both with the bare oculus and under the microscope as single bacterial cells.

Feasible cell counts

As I predicted, the more dilute ( 10-8 ) solution will hold less E.coli bacteriums turning on its surface. As there were 20 different braces making the practical, and the dilutions were all done 20 times by different people, there is plenty room for mistake from taint of inaccurate measurings.

The 10-5 agar home bases had many E.coli bacterial settlements turning on it, harmonizing to the consequences there was far excessively many bacteriums that it was excessively many to number ( TMTC ) . Gradually as the dilution increased the bacterium became less, 10-6 dilution had Numberss runing from 6-140. Obviously with such great difference within what is meant to be the same dilution there was some error/contamination nowadays. The most obvious 1s which had mistake are pair Numberss 18 and 19 there was TMTC throughout ( 10-6, 10-7 and 10-8 ) . What would be expected is that fewer bacteriums should be present in the 10-8.