FAULT IN CULTURE MEDIA

There are varieties of fault found in culture media mostly when preparation of dehydrated media is in progress such as;

• Inappropriate weigh, inaccurate measure of water: this problem take place as a result of faulty calibration of weighing balance, and measuring cylinder. The problem can be solved by making sure that cylinder is well calibrated and weighing balance is in good condition.
• Denaturation of media: this problem occurs as a result of overheating of the media during cooking or sterilization. The problem can be solve by heating the media in an appropriate temperature, that will prevent boiling for several hours.
• Toxicity of the media: these occur as a result of overheating of media that contain carbohydrate, when the media is over heated it form a toxic substance in the media. This can be prevented by not overheating the media.
• Concentration of ingredient in the petri dish: this problem occurs as a result of too much lust of water and condensation. This can be avoided when media is left to cool bellow 49 ºC before pouring into the petri dish
• Media result in their destruction: this occurs as result of addition of heat labile as a supplement i.e. serum or blood after sterilization to the hot media. This can be avoided by allowing the media to cool before adding blood or serum as a supplement to the media.

When media is received make sure that the day of receiving and the day of opening the media are recorded down.

MEDIA PH ADJUSTMENT

It is very important that media should be in an accurate PH value before use. The PH value of media to be use depend on the micro organism to be culture, like some organism growth in an acidic PH, while some growth in a basic PH, so any of these found, PH meter should be used to access the PH when adjusting to the required value. For example when the media is too acidic and need to be adjusted to basic status, NaOH should be used to adjust it the desire value, in reverse if it is too acidic, HCL should be used to adjust it to the desire status. Another method can be used to adjust the PH value, these are manually operated, the method is the used of colorimetric and lovibond comparators techniques.

COLORIMETRIC TECHNIQUE

This method used color indicator to examine the value PH of the media when adjustment is in progress. The indicator is added to the media with other prepared standard solution, the standard solution serve as a control agent when the medium is been adjusted the color of the media is compared with the color of the media until the media color matched with the standard solution that it the point of the desire result.

To carry out this procedure there some apparatus used, this is listed bellow;

1. Comparator rack
2. Set of standard PH tube, i.e. made up of buffer solution and indicator
3. Comparator tube
4. Phenol red as indicator solution
5. 10M and 0.1M HCL, 10M and 1.0M and 0.1M NaOH
6. Micro-burrete
7. Pipette

PROCEDURE

1. Set six comparator tube in a comparator rack, in the first three of the tube add 5ml of the media and add 5ml of water to the remaining three.
2. To the first tube of the media, add indicator as it is present in the standard PH volume

• Make sure that the standard PH used should be bellow and above the required value, for example if the required value is 6.7PH then the standard pH tube to be used will be 6.6pH and 6.8pH value.

1. If the medium is too acidic then add 0.1M NaOH from a burette to fill the color, the color of the tube containing indicator and the medium, but if the medium is too alkaline, then add 0.1 HCL.
2. Measure the volume of acid or alkaline importantly in other to adjust the reaction of 5ml of the media placed in the tube.
3. Take two average reading and calculate the volume of acid or alkaline to be added to the medium and add the require volume of acid or alkali in the concentrated medium.

Mathematical aspect of it is that when 0.5ml of 0.1M HCL is needed to raise the pH of 5ml of the medium, then 2.5ml of 0.1MHCL is needed to raise the pH of 25ml of the medium, these happen continuously depend on the volume of the media. When the alkali or acid is added to the medium, mixed the compound very well and used the same technique explained above to check the pH value of the medium.

LOVIBOND COMPARATOR

This method and the colorimetric technique is the same, it just that there is a little slice different. The sample of the medium together with the indicator in marched against the slide different is that

1. In the first two of the last tube in the comparator rack, is where 5ml of the media is added to, instead of the three which was explained in a precious techniques.
2. Standard volume of indicator is added to the second tube
3. At this point the comparator is close and indicator disc is turn on for pH reading.
4. Add alkali or acid to the first tube till the colors are marched.
5. Calculate the amount of acid or base added to the medium in the way the colorimetric method is calculated above all.

Note: if agar is used the tube that is makes up of agar and indicator with the one contains only agar must be left to cool and solidifies before any other things. Reading must not take place until the media cooled. Before tube is used, they must be washed very well with distilled water. And finally never compare their color in a direct sun light, special view can be used for artificial light.

SUMMARY TECHNIQUE

1. All apparatus which will be used for media preparation must be clean and dry.
2. Estimation of pH value must be carried out by using pH meter, in other to avoid wrong used of pH value media.
3. Fresh sterile or ion free water with appropriate standards must be used. The water must be free from any source of contamination.
4. If dehydrated medium is used, make sure that water is added and stirred, if the media contain agar it should be allowed to stand for at least 30 minute before heating. In care of boiling media contain agar on hot plate care should be taken for the media not to boil and flow out from the flask.
5. In a situation where by the medium is sterilized in a flask, there is a tendency that the volume of the media must surely affect the sterilization that is required. Also it not possible to sterilized volume of a media greater than 2litre in an autoclave without affecting the performance of the media.
6. There are media preparatory which is used all over to prepared media now, in the process media is heated in a close pressure, before heating remember that media must first be stare. When heating and cooling time is consider reduce these pavmedium in the way the colorimetric method is calculated above all.\nNote: if agar is used the tube that is makes up of agar and indicator with the one contains only agar must be left to cool and solidifies before any other things. Reading must not take place until the media cooled. Before tube is used, they must be washed very well with distilled water. And finally never compare their color in a direct sun light, special view can be used for artificial light. All apparatus which will be used for media preparation must be clean and dry.\nEstimation of pH value must be carried out by using pH meter, in other to avoid wrong used of pH value media.\nFresh sterile or ion free water with appropriate standards must be used. The water must be free from any source of contamination.\nIf dehydrated medium is used, make sure that water is added and stirred, if the media contain agar it should be allowed to stand for at least 30 minute before heating. In care of boiling media contain agar on hot plate care should be taken for the media not to boil and flow out from the flask.\nIn a situation where by the medium is sterilized in a flask, there is a tendency that the volume of the media must surely affect the sterilization that is required. Also it not possible to sterilized volume of a media greater than 2litre in an autoclave without affecting the performance of the media.\nThere are media preparatory which is used all over to prepared media now, in the process media is heated in a close pressure, before heating remember that media must first be stare. When heating and cooling time is consider reduce these pave way for large sterilization of media up to 100litre without affecting their performance.\nOn adding a supplement and enrichments, the media should be allowed to cool before adding the substancee way for large sterilization of media up to 100litre without affecting their performance.
7. On adding a supplement and enrichments, the media should be allowed to cool before adding the substance.