Commercial active dried yeast

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Synopsis

This experiment aims to identify the merit of using pour plate method and spread method. The growth of the bacteria will determine the whether the experiment is successful. The validity range will be 30-300 colony forming unit with only one type of colony. The unsuccessful result is when there is no colony form; less or more than the validity range or more than one type of colony formed (cfu).

10 fold serial dilutions are done starting from 10-1 to 10-9. This is the practice for all unknown sample and know concentrated sample. This dilution of 10-6 to 10-8 and 10-7 to 10-9 is plated on both the spread plate and the pour plate with duplicate sets respectively. Incubation stage of 25°C for 2 days after plating and the plate is inverted.

The result for spread plate is only valid for the dilution of 10-6 which is 61 and 50 cfu/ml. As for the pour plate none of the result is valid.

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The cause of the invalid result is because of the dilution techniques. When diluting from higher concentration to a lower concentration, did not change the pipette tip. With this most of the colony count is more than 300.

I conclude that although one of the plate counts is valid for the spread plate. It is not a successful experiment.

Introduction

Purpose

This experiment aims to identify the merit of using pour plate method and spread method.

Background

Active dry yeast is a type of yeast used to raise dough for baking bread, rolls, a few types of cake, and for any type of risen bread. Yeast is essentially tiny, living cells, and the specific yeast used in active dry yeast is Saccharomyces cerivisiae. Yeast is able to raise dough or created leavened dough because it responds to sugar, converting it to carbon dioxide (CO2). For centuries, the most available form of yeast was fresh yeast. While fresh yeast works very well, it has a short shelf life. More commonly you will find active dry yeast, which was first developed during World War II by the Fleischmann Company. The advantage to active dry yeast is that it can be kept in small packages for a year to two years.

Methodology

Colony count methodology is a tool for estimating a microbial population in a sample. Since different organisms have different need, hence suitable nutrients, temperature of incubation and gases may affect the growth of this organism.

Theory

Method to Obtain Pure Cultures

Serial dilution

Dilution is critical as the unknown sample may have more than one cell. By serial diluting an organism in an agar plate can be reached where only one cell remains in the medium. In the end, a pure culture is obtained. Also, from an initially high concentration, the cell concentration is decreased. Cell concentration in a sample can be in a range of thousand and millions and even billions. Therefore, it makes good sense to dilute the sample. One effective method without using too much diluent is by serial dilution. A process of diluting a sample is by performing a series of repeated dilutions. The serial dilution can be in the form of 2-fold, 5-fold, 10-fold or even 1000-fold dilution, depending on the cell concentration of the sample.

Direct method of cell enumeration

Rationale

Plate count for isolation of pure culture is either by pour plate or spread plate. The rationale of this method is to see the viable cells grow to form colonies on the agar plate One colony seen on the agar plate is equal to one cell. The acceptable count of colonies is between 30- 300 colonies per plate.

Pour Plate Method

Pour plate is a common method used in routine testing labs and usually done in duplicates. The molten agar must be cooled to about 50°C before mixing with the organism. If not the organism would be killed. Colonies form using pour plate method is in and on the surface of the agar.

Spread Plate Method

Spread plate is another common method used in a routine labs and is usually done in duplicates. The colonies form on the agar surface.

Procedure

Serial dilution procedures

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10ml valve with 9ml of saline or sterile water. Shake the undiluted active dried yeast. Then inoculate 1ml of active dried yeast and put in the 9ml of saline or sterile water. Shake the solution and label 10-1. Shake the 10-1 and inoculate 1ml of active dried yeast and put in another the 9ml of saline or sterile water. Shake the solution and label 10-2. Shake the 10-2 and inoculate 1ml of active dried yeast and put in another the 9ml of saline or sterile water. Shake the solution and label 10-3 . Shake the 10-3 and inoculate 1ml of active dried yeast and put in another the 9ml of saline or sterile water. Shake the solution and label 10-4 . Shake the 10-4 and inoculate 1ml of active dried yeast and put in another the 9ml of saline or sterile water. Shake the solution and label 10-5. Shake the 10-5 and inoculate 1ml of active dried yeast and put in another the 9ml of saline or sterile water. Shake the solution and label 10-6 . Shake the 10-6 and inoculate 1ml of active dried yeast and put in another the 9ml of saline or sterile water. Shake the solution and label 10-7 . Shake the 10-7 and inoculate 1ml of active dried yeast and put in another the 9ml of saline or sterile water. Shake the solution and label 10-8 . Shake the 10-8 and inoculate 1ml of active dried yeast and put in another the 9ml of saline or sterile water. Shake the solution and label 10-9 .

Plating using Spread Plate

Using 0.1ml of 10-6, 10-7 and 10-8 dilution spread onto duplicate plates of malt agar using a "hockey stick". When feeling frictional force acting, stop the spreading. Incubate the plate for inverted at room temperature (25°C) for 2 days. After incubation, choose only plate that contain between 30-300 colonies per plate and work out the number of yeast cell as cfu/g

Plating using pour plate

Pipette 1ml of 10-7, 10-8 and10-9 dilution into duplicate Petri dish and add 15ml of molten malt agar ( app 45°C) and mix gently and allow the agar to set. Incubate the plate for inverted at room temperature (25°C) for 2 days. After incubation, choose only plate that contain between 30-300 colonies per plate and work out the number of yeast cell as cfu/g

Discussion

Only spread plate of dilution of 10-6 is in range. The rest of the plate of both spread plate and pour plate is invalid. One of the problems is the dilution prepared by me and my partner. The contamination by not changing the pipette tips lead to the over growth of the yeast cells.

Problems of pour plate method

Limitation of the microorganism

Aerobic microorganism may not be suitable as the growth is largely in the agar rather on the surface of the agar.

Longer incubation period

Colonies in the agar may not be visible until later. The colonies in the agar are in partially anaerobic condition. Facultative anaerobes will therefore grow slowly and hence, will not be very obvious after a day of incubation.

Under-estimation of the cell numbers

Some yeast colonies maybe overlapping one another and may be seen as a single CFU. Furthermore the cells are exposed to warm molten agar and this can kill some heat-sensitive yeast and give a lower CFU plate count.

Problem of spread plate

Some yeast colonies maybe overlapping one another and may be seen as a single CFU.

Conclusion

This is a successful experiment although the dilution is cross- contaminated. This is because both the spread and pour plates use the diluted sample which the spread plate shows a valid colony formed. In the future when handling with yeast sample, I will plate the yeast using spread plate method. This is mainly because that yeast is heat- sensitive organism and aerobic fungus.

References

  • Tricia , 2003, what is active dry yeast , 11 January 2020 , http://www.wisegeek.com/what-is-active-dry-yeast.htm