Key Area 1
(f) Aseptic technique & cell culture
Aseptic technique eliminates unwanted microbial contaminants when culturing micro-organisms or cells. Aseptic technique involves the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants.
Aseptic technique involves the use of heat (Bunsen burner required) and chemicals (often ethanol and Virkin) to disinfect surfaces and equipment to be used.
An autoclave is required to sterilise materials and culture before and after use. Steam under pressure is used to produce a temperature of ~121°C for 15minute which kills all microbes, including bacterial endospores (Microbiology Society, 2016).
A microbial culture can be started using an inoculum of microbial cells on an agar medium, or in a broth with suitable nutrients. Many culture media exist that promote the growth of specific types of cells and microbes.
The following video (from start to 2min 40s) shows how an inoculum from nutrient broth can be used to culture bacteria onto an agar medium. Click on the pink box below to watch.
Nutrient broth is the media we tend to use in schools for the growth of a broad spectrum of microbes.
Animal cells are grown in medium containing growth factors from serum. These protein molecules promote cell growth and proliferation and are essential for the culture of most animal cells.
Different growth factors are available to add to culture medium depending on the type of cells being cultured.
Estimating cell numbers in liquid culture by plating out
Plating out a liquid microbial culture on solid media, like the researcher is doing in the left image, allows the number of colony-forming units (CFUs) to be counted and the density of cells in the culture estimated.
The right image shows that a serial dilution (1:1000) of the original culture was performed and 1ml of this was plated out of solid medium. However, the number of CFUs is still too high to count and further serial dilution of the culture is needed to achieve a suitable colony count.
SQA Past Paper Question 3
Try the question below - the answer can be found here.
Reference: SQA (2017), CfE Advanced Higher Biology examination paper, section 1, question 3 (page 3), available at https://www.sqa.org.uk/pastpapers/findpastpaper.htm?subject=Biology&level=NAH [accessed on 02.05.20]
Copyright © Scottish Qualifications Authority
In culture, scientists can study processes use primary cells or tumour (immortilised) cell lines. Primary cells are isolated directly from tissues and retain normal cell structure and functions. This means data obtained using primary cells is more relevant and reflective of the in vivo environment.
Unfortunately, primary cells have a finite lifespan and are extremely difficult to culture. This has led scientists to develop many tumour cell lines that can perform unlimited division and are more resilient in media.
Now is a great time to stick in your air pods and go for a walk, listening to Episode 70 of "This podcast will kill you". This episode is all about HeLa cells - the common link between topics from smallpox to scurvy, vaccines to birth control. These cells have been crucial to the development of technologies and our advancement of knowledge. This episode explores the woman behind the cells, the ethics of using them and what makes them "immortal".
HeLa cells, an immortalised tumour cell line, obtained from Henrietta Lacks in 1950 have been fundamental to many scientific advancements.
Estimating cell numbers in a liquid culture using haemocytometers
A haemocytometer is a graduated microscope slide that can be used to estimate cell numbers in a liquid culture. It was originally designed to estimate the density of blood cells, hence its name. The counting chamber has a known volume so by counting the number of cells in a particular area of the grid, a cell density can be calculated.
Disposable haemocytometers (like the ones shown above) and reusable glass haemocytometers are available for use in your school lab.
Here, a sample of yeast has been stained and loaded into one chamber of the C-Chip haemocytometer for visualisation on a microscope.
The grid in counting chamber of the haemocytometer looks like this when viewed under a microscope.
The microscope is used to focus in on the central grid of the counting chamber.
The number of cells found within this central grid is counted. Since the volume of this chamber can be determined, simple calculations and be performed to estimate the number of cells in the original culture. Let's try an example in the next Task.
Task 13
Cheeky Charlie charmed his way into the class on Monday. He collected his culture from the incubator in the Technician's room and, having added a suitable stain, used a piece of capillary tubing to add a small sample of his culture to a haemocytometer chamber. He carefully adjusted the focus on the microscope until he could see the counting grid in the middle of the chamber. This is (roughly!) what he saw:
Charlie looked up the haemocytometer details and learned that the central square was 0.1cm x 0.1cm, with a depth of 0.01cm.
Based on the cell count from the central sequence, calculate the concentration of animal cells in 1cm3 of the culture.
Hint:
Determine the volume of the central square.
State the number of cells found in the volume of the central square.
Work out how much larger 1cm3 is compared to the volume of the central square and then multiply this value by the number of cells in the central square.
Answers are available here.
Vital Staining to Estimate Viable Cell Counts
Vital staining can be applied to culture to distinguish between living (viable) and dead (non-viable) cells. An example of a vital stain is methylene blue - this stain readily permeates across the membrane of cells but is reduced to a colourless compound by any viable cells (thus viable cells appear colourless when viewed with a microscope). Other vital stains are actively pumped out of living cells but remain inside dead cells - again, the ultimate consequence of this action is that viable cells appear colourless whereas dead cells appear coloured.
SQA Past Paper Question 4
Try the question below - the answer can be found here.
Reference: SQA (2017), CfE Advanced Higher Biology examination paper, section 1, question 1 (page 2), available at https://www.sqa.org.uk/pastpapers/findpastpaper.htm?subject=Biology&level=NAH [accessed on 02.05.20]
Copyright © Scottish Qualifications Authority
Go to SCHOLAR:
1.6 Aseptic technique and cell culture
1.7 Learning Points
1.8 End of topic test
to consolidate and explore your understanding of Key Area 1.
Click here to access a Quizlet on Topic 1, Key Area 1.
Congratulations!
You have now completed Topic 1, Key Area 1 on Laboratory Techniques for Biologists.
Your teacher might now issue you with Learner Check 7 to check your learning of the Topic 1 content so far.
You are now ready to move onto Key Area 2 on Proteins.
