Everytime i found different OD data which was fluctuating and sometimes continue increase of OD .Though growth curve and colony under microscope dont tell wheather it is contaminated or not.If bacterial stock is contaminated with other bacteria, How can be sure that whether it is contaminated or not? .Is there any other method to solve the problem?. Also,The genetic map of this bacteria is not searchable (E.coli MG1655)and i dont want to go to validate with molecular determinants. Please give valuable suggestions.Thanks.
Yes, one can follow the above said methods,
1. One can streak it on a suitable agar media, it depends on what kind of bacteria, whether its a Gram positive or Gram negative (one can use McConkey, Baird parker ).
2. stain the culture to cross verify and to differentiate the cells.
3.Most contaminants could be from genera Bacillus .
Hi there,
To make sure your culture is pure, always streak on plate to get isolated clones from the stock and start culture from these. If you want to determine if your isolated clones are actually what you expect or contaminant you have to get through strain characterization.
Hi,
Go for Gram staining or any specific staining for the bacteria and view under microscope.
Once you isolated the pure culture store it in Selective media or if possible add some antibiotics to which particular bacteria is resistant and other bacteria are susceptible.
1. Perform Gram staining and look at the morphology of the bacterial cells, if contaminated more than one cell type shall be visible.
2. Streak the culture on a suitable agar based medium and observe color and type of cfus.
If Bacillus sp. you are using, heat the culture at 80 C for 10 min and culture on a suitable medium. Most contaminating non sporulating cells will be killed and bacilli survive!
I agree with Dominique, by streaking out for single colonies on a plate you can readily visually determine whether you have contaminants.
Yes, one can follow the above said methods,
1. One can streak it on a suitable agar media, it depends on what kind of bacteria, whether its a Gram positive or Gram negative (one can use McConkey, Baird parker ).
2. stain the culture to cross verify and to differentiate the cells.
3.Most contaminants could be from genera Bacillus .
According to kind of bacteria, you can streak it on a special culture and see colonies.
Re-isolate single cell colonies on suitable agar that support long life for bacteria (low in nutrients, CaCO3 added) after long shacking in water. Grow them for 4-5 days (to obtain slow-growing bacteria) and test at least 50 colonies for morphology and biochemical traits.
But, only 16S rRNA sequencing (at least 500 bp) will give you precise answer.
If bacterial culture is contaminated than we can confirm on seeing the bacterial growth colour because mostly different colour will be seen and we can confirm on staining the bacterial sample by gram staining because under microscope different type cell will be seen .
gram staining, streaking will surely help you. Streaking your culture on selectable agar plates also help you to isolate your own stock original cells.
If the bacterium of your interest is a Bacillus sp., you can head the suspected contaminated broth, a small volume at 55-60oC for about 10 min, the bacillus survived because it produces endospores that are often heat resistant. When you will subculture heated broth, the cells shall form colonies on an Agar plate.
Hi,
If stock contamination is your problem, a streak plating from the stock would be easy to categorize contamination. If not grow them in the flasks, mid-way during the fermentation, collect the sample, streak them and observe them; THese steps would help you to sort out if the contamination is in the stock or if it arose during subsequent stages.
I agree with Alex Ignatov that you would need to do DNA fingerprinting of the 16s rRNA to make positive identification. It is the only reliable way to do so. Kits to do the fingerprinting by PCR are readily available, and here is one article in which the authors describe performing the technique in order to identify different strains of bacteria in a sample: Plengvidhya, V., Breidt, F., Lu, Z., & Fleming, H. P. (2007). DNA fingerprinting of lactic acid bacteria in sauerkraut fermentations. Appl. Environ. Microbiol., 73(23), 7697-7702.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology