Using the Gram Stain to Help Diagnose Meningitis (Labster Lab Manual | P) Flashcards
What is meningitis and what happens if a pt has meningitis?
It is a medical condition where the protective membranes (meninges) that surround the brain and spinal cord become inflamed and compresses the nervous tissues of the central nervous system
How to diagnose meningitis (method of sx collection)?
A lumbar puncture is performed to collect cerebrospinal fluid (CSF) from the spinal canal
What should be done to CSF after being collected from the pt?
CSF should be sent for biochemical and microbiological testing, including Gram stain
What are the causes of meningitis?
1) Bacterial infections [variety of Gram (+) and Gram (-)]
2) Viral infections
3) Fungal infections
4) Parasitic infections
5) Autoimmune disorders
What is bacterial meningitis?
It is considered a medical emergency and requires prompt antibiotic treatment
What are the examples of bacteria that can cause meningitis?
1) Streptococcus pneumoniae [Gram (+)]
2) Neisseria meningitidis [Gram (-)]
3) Haemophilus influenzae [Gram (-)]
What are the types of meningitis that are less severe (compared to bacterial meningitis)?
1) Viral
2) Fungal
3) Parasitic
4) Autoimmune
What is required for viral, fungal, parasitic, and autoimmune types of meningitis?
Less intense or sometimes no treatment
What is Gram stain (Gram staining)?
It is a differential technique that differentiates nearly all bacterial species into 2 large groups
What is the basis of differentiation in Gram staining?
Based on structural differences in the cell wall of bacteria
What is the significance of cell wall (of bacteria)?
1) It is significant because it protects and shapes the bacteria
2) It is involved in including disease in the infected host
3) It can also be a target for certain antibiotics
What are the characteristics of Gram (+) bacteria?
1) They have a thick peptidoglycan layer
2) They retain the primary stain
3) They stain (or appear) purple
What is the function of peptidoglycan layer for Gram (+) bacteria?
It remains the primary stain used in Gram staining
What is the primary stain used in Gram staining?
Crystal violet
What are the characteristics of Gram (-) bacteria?
1) They have a thin peptidoglycan layer
2) They have an outer membrane
3) They don’t retain the primary stain
4) They stain (or appear) pink
Can Gram staining be used to identify specific bacteria?
No, hence, additional microbiologic tests are needed
What are the purposes of Gram staining?
1) The presence of bacteria can be diagnosed or detected
2) The characteristic [either Gram (+) or Gram (-)] of the bacteria can be determined
What is the purpose of knowing the Gram type and morphology of the bacteria present in the sx?
By this, it aids in selecting an initial antibiotic needed to treat the pt
What is the color of Gram (+) bacteria?
Purple
What is the color of Gram (-) bacteria?
Pink
Can light microscope be used to examine a Gram stained slide?
Yes
How many are the reagents (or stains) used in Gram staining?
4
What are the reagents (or stains) used in Gram staining?
1) Crystal Violet (CV)
2) Iodine (I)
3) 95% Ethyl Alcohol (Ethanol)
4) Safranin
What is (and its function) CV?
1) It is the primary stain
2) It is used to color both Gram (+) and Gram (-) bacteria purple
What is the purpose of iodine?
It acts as a dye-fixator (mordant) that fixes the purple stain in both Gram (+) and Gram (-) bacteria
How is the principle of iodine executed?
The Iodine molecules bind to CV molecules via electrostatic forces, forming a large, insoluble Crystal Violet - Iodine complex (CV-I complex)
What is the purpose of ethanol and what is the principle of action brought by ethanol?
1) It is used to decolorize the Gram (-) bacteria
2) It dissolves the outer membrane and disrupts the thin peptidoglycan, allowing the purple CV-I complexes to escape the Gram (-) cell wall
Do Gram (+) bacteria remain purple after alcohol wash? Why or why not?
Yes, because of the presence of their thick and robust peptidoglycan which withstand the alcohol and retain the CV-I complexes
What is safranin (including its purposes and its working principle)?
1) It is used as a counterstain
2) It stains Gram (-) bacteria pink
3) It is a positively charged molecule (like CV) that binds to negative bacterial cell wall structures
What happens to Gram (+) bacteria after the application of all stains?
Gram (+) bacteria are already colored purple by the darker CV and remain purple despite the addition of the pink safranin. Hence, it stains purple
What should be done after addition of each chemical compounds (or stains)?
It is essential to wash the slide with water (after each step)
What is the time duration needed for staining using CV?
60 secs
What is the time duration needed for staining using Iodine?
60 secs
What is the time duration needed for staining using 95% Ethyl Alcohol?
5 - 10 secs
What is the time duration needed for staining using safranin?
45 secs
What are the components of a Gram (+) cell wall?
1) Thick peptidoglycan
2) Techoic acid
3) Cytoplasmic membrane
4) Membrane proteins
What is cytoplasmic membrane [(including its functions and characteristics; Gram (+)]?
1) It surrounds the cytoplasm of both Gram (+) and Gram (-) bacteria
2) It is similar to one found in the mammalian cell membrane
3) It consists mainly of proteins and lipids
4) It regulates the passage of molecules in and out of the bacteria
What are techoic acids [and its functions; Gram (+)]?
1) It provides protection from harmful substances, anchoring the peptidoglycan to the underlying plasma membrane
2) It helps to stabilize the cell wall
Where can large amounts of techoic acids be found?
In the peptidoglycan of Gram (+) bacteria
What is the characteristic of peptidoglycan of Gram (+) cell wall?
It is thick (20 - 80 nm) mesh of repeating subunits containing sugars and amino acids
What is the function of peptidoglycan [Gram (+)]?
1) It provides rigidity
2) It gives the bacteria its specific shape
Give an example of antibiotic that should not be used or taken by pts (when it comes to microscopic examination of bacteria)
Penicillin
What does penicillin do (principle of action of penicillin)?
It inhibits the synthesis of peptidoglycan which causes the bacteria to die
What is the meaning of NAG?
N-acetylglucosamine
What is the meaning of NAM?
N-acetylmuramic acid
What are the 3 primary layers that comprises Gram (-) bacterial cell wall?
1) The cytoplasmic membrane
2) A thin peptidoglycan layer
3) An outer membrane
Where is the peptidoglycan layer of Gram (-) bacteria located?
In the periplasmic space
What are the components of the outer membrane of Gram (-) bacteria?
1) Lipopolysaccharides
2) Porin proteins
What is the cytoplasmic membrane [Gram (-)]?
It surrounds the cytoplasm of Gram (+) and Gram (-) bacteria
What is the function of cytoplasmic membrane [Gram (-)]?
It regulates the passage of molecules in and out of bacteria
Is the cytoplasmic membrane [Gram (-)] is same with those found in mammalian cells?
Yes
What are the components of the cytoplasmic membrane [Gram (-)]?
Mainly:
1) Proteins
2) Lipids
What is the characteristic of the peptidoglycan of Gram (-) bacteria?
It is thin (5 - 10 nm) mesh of repeating subunits
What are the components of the peptidoglycan of Gram (-)?
1) Sugars
2) Amino acids
Where can the periplasmic space in Gram (-) be found?
It lies between the outer membrane and the cytoplasmic membrane
What are the components of the periplasmic space of Gram (-)?
It consists of a gel-like matrix containing a wide variety of proteins that are important for various cell functions
What is the function of the outer membrane of Gram (-)?
1) It protects the bacteria
2) It acts as a barrier towards certain antibiotics
What are the components of the outer membrane of Gram (-)?
1) Porin proteins
2) Lipopolysaccharides
What is the function of porin protein?
To penetrate the outer membrane of Gram (-)
What is the result of the function of porin protein?
It results to formation of pores which allow passive diffusion of hydrophilic molecules between the bacteria and the exterior
What are lipopolysaccharides?
1) These are specific to the outer leaflet of the outer membrane
2) Carries (-) charge
3) Notorious
What is the function of the (-) charge of lipopolysaccharides?
It contributes to maintaining the shape of the bacteria
Why are lipopolysaccharides considered notorious?
Because they have the potential of causing toxic shock in the infected host
Is Gram stain a delicate procedure and requires attention to detail?
Yes
What are the causes of false results acquired in Gram stain?
1) Improper smear prep
2) Staining techniques
What happens if the bacterial smear prepared is too thick?
Gram (-) will appear darker and could be mistaken for Gram (+)
What should be done when it comes to preparation of bacterial smear?
Try to always apply only a single layer of the organism you want to study
What will happen if the bacteria are overheated during fixation?
1) The bacterial cell wall is destroyed
2) Bacteria are not capable of retaining CV
3) All bacteria will result and appear as Gram (-)
What happens if culture smears are expired?
1) If culture smears are too old, the bacterial cell wall might have already broken down, rendering them incapable of retaining the CV stain
2) All bacteria will appear Gram (-)
What happens if decolorizing is frequently done in Gram staining?
It yields false results
What is under-decolorization?
If the alcohol does not penetrate well the bacterial cell wall
What happens if the bacteria is under-decolorized?
1) If the alcohol (decolorizer) is washed away before it had any effect on the cell wall, the CV molecules will not escape the Gram (-) cell wall
2) It will result to appear Gram (+) colored purple after counterstaining with Safranin
What is over-decolorization?
If the alcohol is left for too long on the slide
What happens if the bacteria is over-decolorized?
1) Leaving the alcohol (decolorizer) on the slide for too long disrupts the cell wall of both Gram (+) and Gram (-)
2) Gram (+) can not retain CV, hence, all bacteria appear Gram (-) colored pink after counterstaining with Safranin
What is the purpose of (+) and (-) control?
These are bacterial smears that are used to test if Gram stain was performed properly
What is the reason why the (+) and (-) controls are not as expected?
The stain was not performed properly
What is used on (+) control?
A known Gram (+) bacteria (Staphylococcus aureus is used in the simulation)
What happens if the Gram stain is performed properly [for (+) control]?
The (+) control should appear purple [Gram (+)]
What is used on (-) control?
A known Gram (-) bacteria (Escherichia coli is used in the simulation)
What happens if the Gram stain is performed properly [for (-) control]?
(-) control should appear pink [Gram (-)]
What should be firstly considered when interpreting a bacterial stain?
It is important to know what type of sx you are working with
What is the characteristic of all bacteria in a pure culture?
All bacteria are identical
What are the components of a mixed culture?
It contains different species of bacteria
Give examples of a clinical sx which may include both bacterial and human cells
1) Blood
2) CSF
Do human cells have peptidoglycan?
None, hence, they appear pink in Gram stain
What is the purpose of rigid cell walls of bacteria?
It maintains the definite shape of the bacteria
