Testing on Escherichia coli

Hello everyone! I am sorry about the late posting again. Was doing my project until I forgot about the blog posting until today.

Another common test in our laboratory is the detection of Escherichia coli. From Ying Ying’s first posting, we know that is LTB tubes are positive (gas production), we will have to continue by inoculating a loop of the positive LTB tube into a BGB and a EC tube.

If the food sample is positive for EC tube (gas production), we can conclude that the bacterium is a lactose fermenter. Further tests to confirm whether it is Escherichia coli needs to be carried out.

A loopful of the culture from the EC tube is being streaked onto an Eosin Methylene Blue agar.
Eosin Methylene Blue agar (EMB)
- Consists of Eosin Y and Methylene Blue dyes which inhibit gram-positive bacteria growth and differentiates lactose fermenting and non-lactose fermenting bacteria
- Lactose fermenting bacteria will appear blue-black
- Non-lactose fermenting bacteria will appear colourless and transparent.
- If positive for Escherichia coli, purple colour colonies with green metallic sheen will grow.

E.coli on EMB plate



After incubating for 1 day at 35◦C, the EMB plate is taken out. If positive for Escherichia coli, purification of a single colony for the EMB is done and incubated.

After purification, biochemical tests are being carried out.

Indole test
- If positive for Escherichia coli, a red ring is being formed when Kovac’s reagent is added. This is because Escherichia coli produces tryptophanase which cleaves tryptophan to produce indole and other products. Kovac’s reagent will then react with indole to form a red ring. (Positive indole test)

Methyl Red test
- If positive for Escherichia coli, the methyl red indicator will turn red due to the acids produce by the degradation of glucose, lowering the pH. (Positive MR test)

Voges-Proskauer test
- If positive for Escherichia coli, there will be no change. This is because E.coli does not produce acetyl methyl carbinol which will react with alpha-napthol and potassium hydroxide to produce pink colour after standing for about 1 hour. (Negative VP test)

Simmon’s Citrate test
- If positive for Escherichia coli, there will be growth on the surface of the Simmon’s Citrate slant but no colour change from green to blue.
Gram-stain
- If positive for Escherichia coli: gram-negative bacillus. (pink, rod-shaped)






















Gram stain of E.coli




Results of the biochemical tests are then recorded and compared with the results of the control to check whether it is positive for Escherichia coli.

~Jeremy~

TG01

Biological Indicators

Hi all.

Sterilization Monitoring Using Biological Indicators

In order to ensure that the autoclave machine is functioning properly and sterilizing appropriately, biological indicators will be used. Biological indicators are contains a spore disc inoculated with bacterial spores (Geobacillus stearothemophillus) for steam sterilization and a culture medium, containing a pH indicator, encased in a crushable, glass ampoule. The acid production associated with growth causes a change in colour of the biological indicator. This aids in the detection of growth.

Methods

1. Place one or more vials in the most difficult location to sterilize and run the cycle. Usually at 121°C for 15mins.
2. Seal the cap immediately after retrieving the vials by pressing down firmly until flush with tube.
3. Allow the tubes to cool. Purpose of cooling is to prevent bursting of ampoule due to heat and pressure.
4. Crush the media ampoule by squeezing the sides of the tube.
5. Ensure that the spore disc has been completely saturated with the growth media. If not, gently tap it on a hard surface until disc is totally immersed in the media.
6. Incubate tubes at 55°C for 48 hours, checking at 24hrs interval. A control should be incubated together with those sterilized tubes.

Results

Control: Yellow

Sterilized Vials: Purple

If the vials that were sent for sterilization turn yellow (original colour is purple), it shows that the autoclaving/ sterilization process failed.

that's all! =)

Suat Fang
tg01
0503328G

Overdue answers to all your questions..Sorry.

Eloo guys. Sooooooooooo Sorry that I took such a long time to answer all your questions. I totally forgot about them until Ms Chew reminded me. Sorry again. Below are the answers to the questions. Hope it will help. All your postings are very interesting and I learnt a lot form all the experiences that you guys blog about.

To Randall
For my lab, if there is any contamination, I would just check the microbes responsible for the contamination. If it is a bacterial infection, I simply add sodium hypochlorite (NaClO) before disposing them as general waste. Fungal infections are the most dangerous among all, as the spores are very hardy and could spread easily. Therefore for a fungal infection, the CO2 incubator will be disinfected.
In my lab, there are a lot of frozen cell lines. Lab stuff also frequently freezes cells. Therefore if there were any contamination, the cultures will be disposed into the biohazard bin and new cell line will be thawed.
If there are still contamination occurring, I will culture the media without any cells or add PBS to the media to check if the media contains any microbes.

To Charmaine
Yup I wash twice with PBS so that for the second wash, all the trypsin will be removed from the media. Like I said in my post, washing with PBS for the second time will decrease the possibility for the cells to be resistant towards the trypsin as trypsin is totally removed in the second washing step. Yup I pipette the cells in the centrifuge tube.

To Eugene
I have 8 cancer lines and they all need different types of media. Each media contains different components. Usually I will just go to the webpage where my lab bought the cells and refer to the recommended media for the particular cell line. The media used in my lab are pre-made by the supplier. Then before using the media, I have to add 10% FBS and antibiotics to the media. I will elaborate on which cell line gets which media during my next post.

To Yong Young
For my lab, there are no limits to the number of passages as I am using cancer cell lines. Cancer cell lines are more hardy and have the machinery to over come cell death. (immortal cell lines). But there’s always a but, even though I can subculture as many times I want for cancer cell lines, I have to check for its morphology as they might revert back to the normal, uncancerous state through series of mutations. I havent encountered any mutations though. If such things happen, I have to discard the particular cell and thaw new cells.

To Shu Hui
Trypsin is an enzyme that will help to dislodge the cells that are attached on the surface of the culture flask. It helps to re-suspend those adherent cells from the culture flask. With this, we are able to subculture and count the cells for other experiments or assays.

To Lizzie
For me, if there is a colour change in the media, I will check the culture under the inverted microscope. If there are cells floating (dead cells), I will change the media as soon as possible. Sometimes media colour changes are also due to contamination. Usually, I will change the media once the colour in a way that is very different form the normal colour of the media. For example I will change media if the media turns orange yellow or yellow. I will not change if the media is orangy-red and there are no floating cells found.

To Zahirah
There are other dyes that can be used for cell counting. I will give you an example of one that I researched on. It is know as a Fluorescent dye. This method indirectly counts the number cell by counting the nucleus with the help of a machine known as the NucleoCounter (an integrated fluorescence microscope designed to detect signals from the fluorescent dye). The dye, propidium iodide (PI) bound to the DNA of cell nuclei. The NucleoCounter counts the total cell count and viability of cell samples such as mammalian, yeast, somatic, fish and sperm cells.

To Ye Tun
I will elaborate more on the cell lines I use for my next post.
In my lab, I use the CO2 incubator system. To me manually adding buffers (done in your lab) to maintain the pH of the culture risk more contamination as you have to expose the culture when adding the buffers. In my lab I use ventilated caps for my culture flasks. Which means, I do not need to unscrew the cap a little to allow gasses to exchange in the CO2 incubator thus minimize the risk of contamination. The ventilated caps pores are made specially small so that no microbes could enter.

To Suat Fang
I will elaborate and explain on MTT assay on my next post.


Have fun ppl.

Najib Bin Hamid
(0503217B)

Question and Answer.

hello guys.

shocked to see a post from me right?!
hmm. im here because, on last friday i was asked to answer a qn that was asked by ms chew.
and finally, i've found the answer. (:


Heres the qn:
Why is there detection of the hemoglobion and blood (2 different parameters instead of just one since they are all RBCs) in the urine dipstick analysis?

Brief Answer:
Cause of RBCs/Hb in urine:
Erythrocytes in the blood transport oxygen from the lung to the tissues by binding it to haemoglobin. Excretion of unphysiological amounts of erythrocytes into the urine - i.e. more than about 130.000/24 h - is most often caused by inflammation or other lesions in the urogenital tract. Free hemoglobin originating from lysed erythrocytes (intravascularily or in the sample during storage/transport) may be found in the urine as well. This can be due to a variety of other diseases. Hemoglobin passes into the urine when the binding capacity of the plasma and the tubular re-absorption capacity have been exceeded. This usually occurs with plasma hemoglobin concentrations of around 60 µmol/l.

The interpretation of results: The practical detection limit of the test pad for blood of the Combur-Test® urine test strips is about 5 erythrocytes/µl and for hemoglobin the amount corresponding to 10 erythrocytes/µl. A homogeneous color change indicates the presence of lysed erythrocytes or free hemoglobin in the urine specimen (hemoglobinuria).
Green dots will show up if intact RBCs cells are present on the test paper (hematuria).
Reference range: 0 - 5 erythrocytes/µl

Difference between Hemoglobinuria & Hematuria
Hemoglobinuria is a condition in which the oxygen transport protein hemoglobin is found in abnormally high concentrations in the urine. The condition is often associated with hemolytic anemia, in which RBCs are destroyed, thereby increasing levels of free plasma hemoglobin. The excess hemoglobin is filtered by the kidneys, which release it into the urine, giving urine a red colour.
Hematuria is the presence of blood in the urine. It is a sign of a large number of diseases of the kidneys and the urinary tract, ranging from trivial to lethal.
p.s. Occasionally "hemoglobinuria" is used synonymously, although more precisely it refers only to hemoglobin in the urine.

Interpretation
Transient micro-hematuria
-Heavy physical training or exhausting activities
-Urinary tract infections
-Trauma to the kidneys or urinary tract
Permanent or recurrent micro-hematuria
-Urolithiasis
-Tumours of the kidneys or the urinary tract
-Glomerulonephritis
-Pyelonephritis
Medicines possibly causing micro-hematuria
Phenytoin, rifampicine, danazole, anticoagulants including acetylic salicylic acid, NSAIDs like ibuprofen, cytostatics like cyclophosphamide


Hope it clears the doubt of yall too! (: seeya!
Natalie
TG01