Lab Safety for Bleach Spills

1) When doing any experiments, you need to fill out the COSHH form (Control of Substances Hazardous to Health). In this experiment, you have been working with bleach. What do you need to do under the following conditions?

a) When you come into contact (skin, eye) with bleach

During the laboratory experiment involving bleach, the reagent may come into contact with the body of the student performing the operation. When bleach comes into contact with the skin, great caution should be taken I order not to contaminate the rest of the body or others. The affected area should be sponged with a cloth with the absorbent property to absorb it off the skin. Then the part should be subjected to excess water to clean it off the skin. A rubber glove should be worn when cleaning the affected area so that it doesn’t spread to other body parts. After cleaning the area, both the hands should be cleaned with warm water and a medicated soap to clear off any particles of the bleaching reagent that may have been in the skin or the hands. The gloves should be disposed of and not reused. Should it spill in the eye, the eyes should be sprinkled with too much flowing water to clear off the bleach. This should be done immediately because the bleach will combine with the liquid in the eyes to form an acid which can make a person blind. After splashing water into the eyes, the person should seek medical attention immediately. Warm water should also be used to slowly clean the eyes in order to ensure that much of the chemical is removed before proceeding to the medical officer for further treatment. The chemical is itself poisonous and the cases of being infected should be never be seen in the laboratory. In case a person ingests it accidentally, the person should be given too much water to neutralize the chemical. Medical help should be sought immediately as the product may get into contact with ammonia thereby putting the life of the person in danger. Another measure that can be taken includes giving the person milk in order to vomit. This will help remove the contents which were swallowed. This should be a first aid measure unless told not to do so by a health officer. Milk should also neo be provided in case the person has some symptoms like vomiting.

b) You spill a large amount of bleach in the cabinet

When a large amount of bleach spills over the cabinet, the person carrying out the experiment should first inform the fellow working close to him or her not to come close to the cabinet. He/she should then wear protective equipment such as the gloves and the laboratory coats. The cabinet should be left running in order to for the gases not be inhaled by the students. The bleach is then decontaminated by first removing other equipment that the spill may have spread to and putting them in a safe container within the cabinet. The contaminated apparatus should never be touched by the hands but should be lifted by tongs, forceps etc. The spill is then covered by a spongy material and allowed to absorb it and then an appropriate disinfectant provided in the laboratory is applied. After putting the adsorbent material in the spill area, the cloth should be left for about 20minutes to ensure the whole contents are absorbed. Students should be careful not to inhale the contents of bleach. Therefore, had masks are encouraged to be put on when handling the chemical. A towel is used to wipe out the remaining fluids in the area to ensure it is dry. The procedure is then repeated by disinfecting the area and carefully wiping the area off for it to become dry. This will ensure that there are no pills left in the cabinet that may be harmful to other people. The apparatus which had the pills are also subjected to disinfection measures then dried. Caution should be taken when disinfecting the apparatus so that nobody contamination occurs.

2. You are using B16 cells, where are they derived from?

B16 cells are tumor cells which have been developed for the study of human skin cancer. Skin cancer has been on the rise over the years and hence the need to develop a model which can be used in the study for cancer. B16 cells have been effective in many types of research such as metastasis and the formation of solid tumors in the human skin. The model was developed from the epithelia of a mice which have almost the same features as that of a human being. The melanin producing organ is suggested due to its network to the lungs and liver thereby providing good metastatic information.

3. When trypsinizing cells, why do we carry out a brief pre-treatment with trypsin?

In trypsinizing, a pre-treatment is first carried out with the trypsin in order to digest the proteins present in the in the culture. Proteins such as FBS are being digested in order for the trypsinizing process to be accurate. The second treatment is the one which the practice is carried out as it detaches the cells through enzymatic digestion from the proteins that are used for the anchoring processes. Therefore, the pre-treatment test is recommended in ensuring the anchoring proteins are digested and later undigested in the process. The proteins act as a catalyst which only speeds up the reaction process but remains in their original state at the end of the reaction. The enzymatic catalysts are important as they bridge the reaction process and ensures the results are achieved within a shorter time.

Take a deeper dive into Examine one Scene from Either of the Films We Have with our additional resources.

4. In a similar experiment, we obtain viable cell counts of 49, 24, 27, 32, 36, 43, 55, 40, 46, 34 and total cell counts of 51, 28, 31, 37, 39, 48, 57, 41, 48 and 34 for each of the 10 fields. For conducting an MTT assay, if you want to make 50mls of a 6.5*104 cells per mls suspension, how much stock solution do we need to add to how much medium?

In the table above, the number of active cells = 386

The number of Total cell count = 414

The number of dead cells = 28

1ml of solution contains = 6.5*104 cells

50mls of solution =?

= (50mls * 6.5*104) = 3.25*106 cells

Therefore, in 50mls of a solution, number of cells to 3.25*106 per ml.

The cell viability in the above experiment = (386/414) = 0.932 or 93%

Therefore, the concentration of stock needed = 3.25*106*0.932 = 3.029*106 cells per ml

5. If you have 25.0mls of cell suspension at 2.14*106 cells per ml, how much of this stock solution do we need to add to how much medium to make the following cell suspensions:

20.0mls of a 4.5*105 cells per ml suspension;

If 25mls of cell suspension at 2.14*106 per ml

1ml represents?

(1*2.14*106)/25 = 8.56*104cells per ml

For 20mls of 4.5*105cells per ml = (4.5*105)/20 = 2.25*104

Therefore, the amount of solution to be added = (8.56*104)/ (2.25*104) = 3.80 cells per ml

15.0mls of 8.9*105 cells per ml suspension;

For 15.0mls of 8.9*105 cells per ml = (8.9*105)/ (15.0) =5.93*104

Therefore, the amount to be added = (8.56*104)/ (5.93*104) = 1.44 cells per ml

60.5mls of 3.15*104 cells per ml suspension;

For 60.5mls of 3.15*104 cells per ml = (3.15*104)/ (60.5) = 5.20*102

Therefore, the amount needed is = (8.56*104)/ (5.2*102) = 1.64*102 cells per ml

25.0mls of 77.7*104 cells per ml suspension (20mks) – please show working and use a sensible number of decimal places

For 25.0mls of 77.7*104 cells per ml = (77.7*104)/ (25.0) = 3.11*104

The amount to be added is = (8.56*104)/ (3.11*104) = 2.75 cells per ml

6. Using a similar format as used for the SOPs in the cell culture handbook, write an SOP for counting cells using a haemocitometer including results from the practical for both plates A and B as worked examples of how to do the calculations.

Standard operating system (SOP) for counting cells using a haemocitometer involves finding the average for each set of counts of both total number of cells and live cells. Plate A and Plate B both represents the averages for all the live cells and the total number of cells.

Cell viability = number of live cells divided by the number of total cells

For plate A = (21/30) * 100 = 70%

For plate B = (55/57) * 100 = 96.5%

In the practical above, the cell viability for both the plates was calculated. This is done by having different counts of cells per plate at a given time than finding the average. This will provide the number of dead cells and the living cells at the time of the experiment. Therefore, the specimen used for analysis should be pure and no contamination should be on the slide as it will give false information. The magnifying lens power should be noted so that the total number of cells found can be converted to the resolution power.