Protistiary - Picking and Cleaning

<%@LANGUAGE="JAVASCRIPT" CODEPAGE="1252"%> Protistiary - Picking and Cleaning

PROTOCOLS Picking and cleaning to make pure cultures
The process of isolation and cleaning protozoa can be achieved in a variety of ways, such as using gravity or phototaxis to draw the selected species through axenic or anti-biotic laced fluids, monitor 'rough cultures' (i.e. material from source that has been allowed to stand in the laboratory for a while with or without some food) and make subcultures when the target species are abundant. However, the most usual technique is to pick cells from their environment with a fine pipette and wash them through several rinses of clean water to remove contaminating species. This is done using a dissecting microscope, target protists identified, and removed WITH THE MINIMUM OF CONTAMINATION. The cells can then be transferred to a slide with some clean (or sterilized or laced with antibiotics water) washed in it, and moved to another slide, eventually being placed in a small dish with a small amount of clean medium and nourishment, and allowed to grow. The objective is usually to make a culture that contains only one species of eukaryote. If sterile (axenic) cultures are required - that is without bacteria - often a lot more trial and error will be required. Antibacterial agents are usually added to the final medium to inhibit bacterial growth or to kill the bacteria. The dissecting scope has great value in the laboratory. Trying to make cultures without one is painful. You will need Dissecting scope Slides Depression dishes (watch glasses) or other small culture vessels Petri dishes to hold depression slides Source water Clean syringes 0.2 µm Nucleopore filter Fine drawn pipettes teats Food (for the protists) Medium for the protist - source water filtered through a 0.2 µm Nucleopore filter usually works, although predatory species will need food. Technique
Assuming that you wish to not only select cells but initiate a clean culture, the normal process is to observe a sample with a dissecting microscope, identify the cells that you wish to remove, and pick those cells out WITH THE MINIMUM OF CONTAMINATION. The cells can then be transferred to a slide with some clean (or sterilized or laced with antibiotics water) washed in it, and moved to another slide, eventually being placed in a small dish with a small amount of clean medium and nourishment, and allowed to grow. You will find that when you are picking cells, it is helpful to have an uncluttered bench and to have the key items, source culture, clide, medium, and culture vessel close to each other.	Typical dissecting scope. One with light coming from below is essential, - top lit illumination will not be satisfactory. Image and copyright M. Richlen.
Put some source water into the syringe and add 0.2 micron filter. This will be used to make sterile water for washing or for cultures.	Syringe with medium and filter. Use clean ones for each sample. Image and copyright M. Richlen.
Note that 0.2 µm Nuclepore filter will usually create a sterile medium but they are not guaranteed to do so. Some smaller bacterial may penetrate through the filters, filters may be cracked or otherwise let organisms through. Larger pore sizes can be selected if you wish to let bacteria through but not protozoa (1 µm filters will usually produce protist free cultures).	0.2 µm filter attached to a syringe. Image and copyright M. Richlen.
Freshly drawn fine pipettes. If you wish to achieve sterile cultures, the pipettes would be sealed at one end with cotton wool and the fine ends of the pipettes would be fused and broken only prior to use. Image and copyright M. Richlen.	Typical glass slides. Use clean ones. Image and copyright M. Richlen.
Put a milliliter or so of clean medium onto a clean slide. Using the dissecting scope, pick out some target cells, replace the source dish with the slide and add the cells to one side of the drop. You will find this easiest if you keep the pipette in more or less the same place, and have the other items you need arranged close to the microscope. If your target species swims, try not to shake the slide. Watch the cells, and as the most mobile reaches the other side of the drop, pick some out with a clean pipette. And repeat the process. If the cells are not mobile, add a small number to the center of the drop, swirl the slide to mix the water and then pick out the dispersed cells.	Selecting protists. Image and copyright D. Patterson.
How to suck items into a pipette. Some folk use a tube that goes to the mouth and some just use a teat. Teats vary enormously in quality, and we prefer the red ones from Agar Scientific, 66a Cambridge Road, Stansted, Essex, UK because they are stuff and rarely leak. As you pick up liquid, pull up the minimum that you need. The aperture of the pipette should be about twice the diameter of the target organism - much wider and you will draw up too much liquid. They are supposed to be available through Pelco in the US - Some types of teats - the one in the middle is the best. . Use clean ones. Protozoa do not like to be transferred, may will commit hari-kiri by blasting themselves against the glass of the pipette. Expect losses of at least half of the cells. Most cells do not like to be transferred to new media, and the level of ‘shock’ can be reduced by using the source liquid as the basis of the medium. Image and copyright M. Richlen.	Mouth pipetting, the pipette is placed in a tube and suctioon is controlled by the mouth. Image and copyright M. Richlen.
When the cells have been picked, prepare a multiwell dish for the initial culture step. Add half a milliliter or so of medium to each depression with any required food. While it is quite easy to find plastic multiwell dishes , they are rarely suitable for these purposes - being too big and with vertical sides it is hard to get the pipette to go where you need it to go. You really need transparent dishes with a curved depression. . You can use slides designed for hanging drops , solid watch glasses, 3 well or 9 well multidepression dishes. All of these are expensive and are re-used.	Multiwell dish. Image and copyright M. Richlen.
When the cells have been added to the medium, the dish can be placed in a larger container, such as a petri dish, on a pad of damp tissue paper. This is to prevent evaporation. Add lid. Check the wells after an hour or so - if the cells survive the first hour they will survive for a longer time. Then check daily for growth. When the cells seem to be growing enthusiastically, move to a larger container increasing the amount of medium. Check as soon as the culture seems to be thriving for any contaminants.	. Multiwell dish in a petris dish with a pad of damp tissue paper. Image and copyright M. Richlen.
Protistiary home page Top of Protocols

PROTOCOLS

Picking and cleaning to make pure cultures

The process of isolation and cleaning protozoa can be achieved in a variety of ways, such as using gravity or phototaxis to draw the selected species through axenic or anti-biotic laced fluids, monitor 'rough cultures' (i.e. material from source that has been allowed to stand in the laboratory for a while with or without some food) and make subcultures when the target species are abundant.

However, the most usual technique is to pick cells from their environment with a fine pipette and wash them through several rinses of clean water to remove contaminating species. This is done using a dissecting microscope, target protists identified, and removed WITH THE MINIMUM OF CONTAMINATION. The cells can then be transferred to a slide with some clean (or sterilized or laced with antibiotics water) washed in it, and moved to another slide, eventually being placed in a small dish with a small amount of clean medium and nourishment, and allowed to grow.

The objective is usually to make a culture that contains only one species of eukaryote. If sterile (axenic) cultures are required - that is without bacteria - often a lot more trial and error will be required. Antibacterial agents are usually added to the final medium to inhibit bacterial growth or to kill the bacteria.

The dissecting scope has great value in the laboratory. Trying to make cultures without one is painful.

You will need

Dissecting scope
Slides
Depression dishes (watch glasses) or other small culture vessels
Petri dishes to hold depression slides
Source water
Clean syringes
0.2 µm Nucleopore filter
Fine drawn pipettes
teats
Food (for the protists)
Medium for the protist - source water filtered through a 0.2 µm Nucleopore filter usually works, although predatory species will need food.

Technique

Assuming that you wish to not only select cells but initiate a clean culture, the normal process is to observe a sample with a dissecting microscope, identify the cells that you wish to remove, and pick those cells out WITH THE MINIMUM OF CONTAMINATION. The cells can then be transferred to a slide with some clean (or sterilized or laced with antibiotics water) washed in it, and moved to another slide, eventually being placed in a small dish with a small amount of clean medium and nourishment, and allowed to grow.

You will find that when you are picking cells, it is helpful to have an uncluttered bench and to have the key items, source culture, clide, medium, and culture vessel close to each other.

Typical dissecting scope. One with light coming from below is essential, - top lit illumination will not be satisfactory. Image and copyright M. Richlen.

Put some source water into the syringe and add 0.2 micron filter. This will be used to make sterile water for washing or for cultures.

Syringe with medium and filter. Use clean ones for each sample. Image and copyright M. Richlen.

Note that 0.2 µm Nuclepore filter will usually create a sterile medium but they are not guaranteed to do so. Some smaller bacterial may penetrate through the filters, filters may be cracked or otherwise let organisms through. Larger pore sizes can be selected if you wish to let bacteria through but not protozoa (1 µm filters will usually produce protist free cultures).

0.2 µm filter attached to a syringe. Image and copyright M. Richlen.

Freshly drawn fine pipettes. If you wish to achieve sterile cultures, the pipettes would be sealed at one end with cotton wool and the fine ends of the pipettes would be fused and broken only prior to use. Image and copyright M. Richlen.

Typical glass slides. Use clean ones. Image and copyright M. Richlen.

Put a milliliter or so of clean medium onto a clean slide. Using the dissecting scope, pick out some target cells, replace the source dish with the slide and add the cells to one side of the drop. You will find this easiest if you keep the pipette in more or less the same place, and have the other items you need arranged close to the microscope. If your target species swims, try not to shake the slide. Watch the cells, and as the most mobile reaches the other side of the drop, pick some out with a clean pipette. And repeat the process.

If the cells are not mobile, add a small number to the center of the drop, swirl the slide to mix the water and then pick out the dispersed cells.

Text Box:

Selecting protists. Image and copyright D. Patterson.

How to suck items into a pipette. Some folk use a tube that goes to t

he mouth and some just use a teat. Teats vary enormously in quality, and we prefer the red ones from Agar Scientific, 66a Cambridge Road, Stansted, Essex, UK because they are stuff and rarely leak. As you pick up liquid, pull up the minimum that you need. The aperture of the pipette should be about twice the diameter of the target organism - much wider and you will draw up too much liquid. They are supposed to be available through Pelco in the US - Some types of teats - the one in the middle is the best. . Use clean ones. Protozoa do not like to be transferred, may will commit hari-kiri by blasting themselves against the glass of the pipette. Expect losses of at least half of the cells. Most cells do not like to be transferred to new media, and the level of ‘shock’ can be reduced by using the source liquid as the basis of the medium. Image and copyright M. Richlen.

Mouth pipetting, the pipette is placed in a tube and suctioon is controlled by the mouth. Image and copyright M. Richlen.

When the cells have been picked, prepare a multiwell dish for the initial culture step.

Add half a milliliter or so of medium to each depression with any required food.

While it is quite easy to find plastic multiwell dishes , they are rarely suitable for these purposes - being too big and with vertical sides it is hard to get the pipette to go where you need it to go. You really need transparent dishes with a curved depression. . You can use slides designed for hanging drops , solid watch glasses, 3 well or 9 well multidepression dishes. All of these are expensive and are re-used.

Multiwell dish. Image and copyright M. Richlen.

When the cells have been added to the medium, the dish can be placed in a larger container, such as a petri dish, on a pad of damp tissue paper. This is to prevent evaporation. Add lid. Check the wells after an hour or so - if the cells survive the first hour they will survive for a longer time. Then check daily for growth.

When the cells seem to be growing enthusiastically, move to a larger container increasing the amount of medium. Check as soon as the culture seems to be thriving for any contaminants.

Multiwell dish in a petris dish with a pad of damp tissue paper. Image and copyright M. Richlen.

Protistiary home page Top of Protocols