Exercise 1: Importance of Aseptic Techniques Flashcards
Explain the term ‘Aseptic technique’
Aseptic techniques are those which attempt to limit contamination of species and infection by pathogenic agents.
Give examples of Aseptic techniques used in a laboratory setting
- Washing your hands before entering the lab, and after leaving
- Flaming inoculating loops before and after use
- Culture media must be sterilized after preparation
- Discarding petri dishes, bottles, tubes, flasks and swabs into their designated disposal containers
- Decontaminating lab benches using a disinfectant agent (eg Alcohol)
- Keeping sources of contamination off the bench (eg Mobile phones)
Why were the NA and MEA plates incubated at two different temperatures?
Since different organisms grow at different temperatures and conditions. Generally speaking, environmental flora will favour growth at 25 degrees celcius (room temperature), whilst flora from within the body will tend to grow at 37 degrees celcius.
What is the role of the MEA plate?
- Is quite acidic
- Is ideal for the isolation of yeasts and moulds
What types of microorganisms would you expect to grow on NA and HBA plates in Part B (Upper respiratory tract)?
Normal flora from the upper respiratory tract:
- Staphylococcus epidermis
- Streptococcus pnemoniae
- Staphylococcus aureus
- Haemophilus influenzae
What types of organisms would you expect to grow on MSA (mannitol salt agar)?
- Selective media for isolation of presumptive pathogenic gram-postive staphylococci
- Selective and differential by the addition of mannitol and phenol red
- Staphylococcus aureus ferments the mannitol to produce acidic end products -> these change the medium from pink to yellow, yellow colonies are formed
Describe the catalase test
- Is used to detect catalase
- Catalase is a haem-containing enzyme responsible for the decomposition of hydrogen peroxide
2H2O2 -> 2H2O + O2 - Several drops of H2O2 are added to a thick suspension of cells on a microscope slide
- Catalase activity is indicated by the appearance of bubbles of gas (oxygen) within a minute
*Media containing blood (eg HBA) may give false-positive reactions as red blood cells contain catalase
Describe the BactiStaph test and the organism it identifies
- BactiStaph test detects Staphylococcus aureus
- Is typically performed after a gram stain and catalase test
- Utilises protein coated latex particles capable of detecting Clumping factor and Protein A in S. aureus
POSITIVE RESULT -> agglutination of black latex suspension
Negative result -> absence of agglutination
What types of organisms could be present on your mobile phone?
- From hand: Staphylococcus epidermis, Escherichia coli (if hands were not washed after going to the bathroom)
- From upper respiratory tract: Staphylococcus epidermis, Streptococcus pnemoniae, Staphylococcus aureus
What steps can you suggest to minimise the level of microorganisms on your mobile phone?
- Wash your hands (particularly after going to the bathroom)
- Cleaning the surface of the mobile phone (using sterile wipes)
Describe what the gram stain differentiates between and how this occurs
- The gram stain differentiates between gram positive and gram negative bacteria
Gram positive:
- Stain PURPLE
- Have a thicker peptidoglycan layer in their cell wall (20-80nm thick)
- Crystal violet and iodine forms a complex in the peptidoglycan which causes these bacteria to stain purple
Gram negative:
- Stain RED
- Have a thiner peptidoglycan layer (2-7nm thick)
- After the purple is decolourised from these bacteria by the 95% alcohol, the carbol fuchsin counterstain causes them to appear red.
Describe the steps involved in performing a gram stain
Prior to beginning the stain, the culture is heat fixed onto a microscope slide.
- Cover the smear with crystal violet (primary stain) and leave for 30 seconds
- Wash off excess stain with water
- Flood the slide with Gram’s iodine solution and leave for 30 seconds
- Wash off excess stain with water
- Decolourise with 95% alcohol for 5 seconds
- wash with water
- Cover the smear with dilute carbol fuchsin (counterstain) and leave for 30 seconds
- Wash with water, drain and carefully blot dry
Describe what the gram stain differentiates between and how this occurs
- The gram stain differentiates between gram positive and gram negative bacteria (on the basis of their cell wall structure)
- It enables the stain, shape and arrangement of bacteria to be visualised
Gram positive:
- Stain PURPLE
- Have a thicker peptidoglycan layer in their cell wall (20-80nm thick)
- Crystal violet and iodine forms a complex in the peptidoglycan which causes these bacteria to stain purple
Gram negative:
- Stain RED
- Have a thiner peptidoglycan layer (2-7nm thick)
- After the purple is decolourised from these bacteria by the 95% alcohol, the carbol fuchsin counterstain causes them to appear red.
Describe the different types of haemolysis
- Alpha haemolysis - refers to partial haemolysis of the red blood cells, where the agar under and around the colonies turns green
- Beta haemolysis - refers to the complete lysis of red blood cells; this is observed as a complete clearing of the medium around the colonies
- Gamma haemolysis - refers to the absence of haemolysis; the medium under and around the colony is unchaged
