Basic Microorganism and Bacteria (L1 and L2)

Order of gram staining

Fixation → crystal violet → iodine treatment → depolarization → counter stain

What is primary stain in gram staining

Crystal violet

What is responsible for determining gram +/- in gram staining procedure

Decolarization

What color is gram positive and why

Purple due to the thick peptidoglycan layer

What color is gram negative and why

pink due to the thin peptidoglycan layer

Bright field microscopy

Source of illumination is visible light to visualize bacteria due to contrast difference in bacteria and medium

Dark field microscopy

Reflected light is used rot visualize bacteria too thin to be seen by bright field

Acid fast positive bacteria

Needs a carbon fuchsin stain (will appear pink) due to the thick mycotic acid it cannot do gram staining

Staph

Clusters

*Gram positive

Strep

Chains

*Gram postiive

Diplo

Pairs → kidney beans or lancet shapes

*Gram negative

Bacillus

Rod shaped

Cocci

Round shape

Arrangement of cocci

Staph, strep, and diplo

Arrangement of bacillus

Chain, single, short rod, club shaped rod, or thin-branching rod

Bipolar staining

Always gram-neg, rod, and singular bacteria

Also known as safety pin appearance

Helicoidal

Spiral shape and requires a dark filed microscope

Pleomorophic bacillus

varies in size and shape

Components of a bacterial envelope

Capsule, cell wall (peptidoglycan), and cell membrane (cytoplasmic membrane)

Bacterial chromosome

singular, circular, dsDNA

Plasmids

extrachromosomal elements, ribosomes 70S

Structures found outside the peptidoglycan/ cell wall

Flagella, pili, and capsule

Function of flagella

motility and chemotaxis, contains H antigen that’s useful in serological identification of bacteria

Common pili function

adherence, motility, and antiphagocytic

Sex pili function

Conjugation (horizontal gene transfer)

function of the capsule / slime layer (k antigen)

Exopolysacchride (repeating units) of…

• single sugar molecules

• Complex polysaccharides

• D-glutamic acid

Medical significance of capsules

They can adhere to the host tissue, you can use the k antigen for serum identification, and for protection in antibiotics and serum factors

Unique component of gram positive bacteria cell wall

Teichoic acid

Unique component of gram negative cell wall

LPS and thick outer membrane

Function of peptidoglycan / cell wall

Interacts with the environment

• Virulence and toxicity factors

• O antigen

• Anchors bacteria’s external surface

Site of differential staining occurs

Shields against environmental changes

• Prevents cell bursting by osmotic forces

Murein structure

Structure of the peptidoglycan → fabric shell/ mesh like structure that encases the cell

alternating network of monosaccharide subunits that are connected through short peptide chains

What supports the mechanical strength of peptidoglycan?

The difference in the amino acid side chains, cross linking creating rigidity, and the alternating NAM and NAG

What is peptidoglycan composed of

NAG and NAM that alternate → NAM contains amino acid side chains and therefore is more structurally important

Outer wall layer structure of gram negative bacteria

Lipid bilayer, proteins, and LPS (endotoxin)

Proteins and lipoproteins found on the outer wall layer of gram negative bacteria

pore protein (PP), ompA protein (A), lipoprotein (LP), and nutrient binding protein (BP)

Function of the outer wall layer of gram negative bacteria

lipid bilayer for differential permeable only to hydrophobic substances

Core sugar in gram negative cell wall

KDO

Properties of LPS

Found in the outer membrane, highly specific to O antigen which is critical for maintaining structure, does not form toxoids, effects many organ systems (broad specificity), heat stable

SIRS

clinical manifestations such as increased body temperature, tachycardia, tachypnea, and leukocytosis

Clinical manifestations of LPS

Different types of sepsis

Sepsis

SIRS + proven infection

Severe sepsis

Sepsis + organ failure

Septic shock

Severe sepsis + refractory hypotension

Distributive shock

warm shock, hypovolemic shock → due to LPS activate macrophages and binding to hypothalamus

• fever, chills, leukopenia, tachycardia, tachypnea, hypotension, shock

LPS detection

Limulus amebocyte lysate test (LAL test) or monoclonal antibodies

Immune effects of peptidoglycan (gram pos)

induces TNF-a and IL-6 causing hypotensive shock → similar to LPS

Function of Lipoteichoic acid (gram pos)

adhesion and antiphagocytic

Function of teichoic acid (gram pos)

Activates complement and can initiate a hypotensive shock

PAMPs

induce specific transaction pathway if recognized by immune system

located on peptidoglycan, teichoic acid, LPS, CpG nucleotides, and n-formyl methionine-leu-phenylalanine

Endospore formation

Due to adverse condition for bacteria survival (heat and chemical resistant), can remain dormant for decades until favor conditions are present

Examples of atypical bacteria

Contain mycotic acid → basis for acid-fast stain

Mycobacterium and Nocardia

Mycoplasma

Lacks peptidoglycan and the cell membrane is stabilized by sterols (wall less pleomorphic bacteria)

Chlamydia

Have gram negative cell wall architecture but minimal peptidoglycan

Components of LPS

O antigen, KDO, and Lipid A