Staining & Culture Media, Medical & Surgical Asepsis

• Staining


  To facilitate visualization, meant to give color to organisms, making them easier to see under the microscope
• Simple stains


  • Use of a single dye, either aqueous or alcohol-based
  • Used to visualize cell shape, size, and arrangement of bacteria
  • Stains give up or accept hydrogen ion, leaning the stain positively charged
• Differential stains


  • Used to differentiate one group of bacteria from another
  • Gram stain
  • Acid-fast stain
• Gram stain


  1. Distinguishes gram-positive bacteria from gram-negative bacteria

  2. Gram-positive bacteria stain blue or purple

  3. Gram-negative bacteria stain red or pink

  4. All cocci are gram-positive except Neisseria, Veillonella, and Branhamella

  5. All bacilli are gram-negative except Corynebacterium, Clostridium, Bacillus, and Mycobacterium
• Safranin


  Counterstain or secondary stain
• Acid-fast stain


  1. Used for bacteria with high lipid content in their cell wall, hence cannot be stained using Gram stain

  2. Ziehl Neelsen Stain (hot method)

  3. Kinyoun Stain (cold method)
• Carbol fuchsin


  Primary stain
• Methylene blue, Malachite green


  Counterstain or secondary stain
• Special stains


  • Used to demonstrate specific structure in a bacterial cell
  • Loeffler Alkaline Methylene Blue (LAMB)
  • Hiss Stain
  • Dyer Stain
  • Fischer-Conn Stain
  • Dorner and Shaeffer-Fulton Stain
  • India ink or nigrosine
• Culture media


  An aqueous solution to which all the necessary nutrients essential for the growth of organisms are added
• Classification of culture media

  • According to physical state (liquid, semi-solid, solid)
  • According to chemical composition (synthetic, non-synthetic)
  • According to functional type (general purpose, enriched, selective, differential, transport, anaerobic)
• Liquid media


  • Called broths, milk, infusion
  • Suited for the propagation of many organisms, fermentation studies, and other tests
• Semi-solid media


  • Exhibit a clot like consistency at ordinary room temperature and contain agar at concentration of 0.5% or less
  • Suited for culture of microaerophilic bacteria or for the study of bacterial motility
• Solid media


  • Contain a solidifying agent such as 1.5%-2% agar
  • Used for isolation of bacteria and fungi or for determining the colony characteristics of the organism under study
  • Can either be liquefiable (or reversible) solid media or non-liquefiable (or non-reversible) solid media
• Synthetic media


  • Contain chemically defined substances which are pure organic and/or inorganic compounds
  • Precise chemical composition of a synthetic medium is known
• Non-synthetic media


  • Complex media that contain at least one ingredient that is not chemically defined
  • Most are extracts of animals, plants, or yeasts
  • Can support the growth of more fastidious organism
• General purpose media


  • For primary isolation of a broad spectrum of microbe
  • Support the growth of both pathogenic and non-pathogenic organisms
  • Peptone water nutrient broth, and nutrient agar
• Enriched media


  • Contain complex organic substance such as blood, serum, or special growth factors
  • Designed to increase the number of desired microorganisms without stimulating the rest of the bacterial population
  • Blood agar
  • Chocolate agar
• Selective media


  • Contain one or more substances that encourage the growth of only a specific target microorganism and inhibit the growth of others
  • Thayer Martin agar
  • Mannitol Salt agar
  • MacConkey's agar
  • Lowenstein Jensen medium
  • Saborauds's Dextrose agar
• Differential media


  • Designed to show visible differences among certain groups of microorganisms
  • Differences may be in the form or variations in colony size or color, changes in color of culture media, or formation of precipitation or gas bubbles
  • Allow the growth of more than one target microorganism that demonstrate morphologic variations in colony morphology
• Transport media


  • Used for clinical specimens that need to be transported to the laboratory immediately after collection
  • Prevent the drying of specimen and inhibit the overgrowth of commensals and contaminating organisms
  • Charcoal is added to neutralize inhibitory factors
• Anaerobic media


  • Used specifically for organism that cannot survive in the presence of oxygen and require reduced oxidation reduction potential and other nutrients
  • Supplements with nutrients such as vitamin K and hemin
  • Undergo boiling to remove dissolved oxygen
• Asepsis


  Refers to a condition in which the individual and his/her surrounding environment are free of any microorganisms
• Sepsis


  Refers to the clinical condition where an individual develops a systemic reaction to a bacterial infection that starts from a localized infection in one part of the body
• Goals of asepsis


  • Protection of the patient from the hospital acquired or nosocomial infections
  • Prevention of the spread of pathogenic microorganisms
• Factors in infection occurrence of patients

  • Suppression of the immune system
  • Prolonged duration of illness
  • Procedures that patients undergo in the healthcare facility
• Common pathogenic microorganisms


  • Escherichia coli
  • Staphylococcus aureus
  • Pseudomonas aeruginosa
  • Candida albicans
  • Enterococcus
• Primary locations of infections

  • Surgical wounds
  • Urinary tract
  • Respiratory tract
  • Bloodstream
• Chain of infection


  • Pathogen (bacteria, virus, fungi, parasite)
  • Reservoir (people, animals, soil, food, water)
  • Portal of exit (coughing/sneezing, body secretions, feces)
  • Mode of transmission (direct contact, indirect contact, vectors)
  • Portal of entry (mouth, nose, eyes, cuts in skin)
• Asepsis


  The absence of disease producing microorganisms
• Factors in infection occurrence of patients

  • Suppression of the immune system
  • Prolonged duration of illness
  • Procedures that patients undergo in the healthcare facility
• Common pathogenic microorganisms


  • Escherichia coli
  • Staphylococcus aureus
  • Pseudomonas aeruginosa
  • Candida albicans
  • Enterococcus
• Primary locations of infections

  • Surgical wounds
  • Urinary tract
  • Respiratory tract
  • Bloodstream
• Chain of infection


  • Pathogen (bacteria, virus, fungi, parasite)
  • Reservoir (people, animals, soil, food, water)
  • Portal of exit (coughing/sneezing, body secretions, feces)
  • Mode of transmission (direct contact, indirect contact, vectors)
  • Portal of entry (mouth, nose, eyes, cuts in skin)
  • Susceptible host (elderly, infants, immunocompromised, anyone)
• Medical or clean asepsis

  Refers to the absence of disease producing microorganisms, aimed to decrease the number of organisms and prevent their spread in the general clinical setting
• Surgical or sterile asepsis

  Absence of all microorganisms, aimed to eliminate microorganisms from an area in the body where surgical procedures will be performed
• General aseptic procedures


  • Frequent handwashing of hospital personnel
  • Prompt and safe disposal of contaminated materials
  • Regular checking and emptying of containers for surgical drains
  • Prompt cleaning of soiled or moist areas
  • Proper labeling of containers regarding the date and time of disposal
• Purposes of handwashing


  • To reduce the flora on the healthcare worker's skin
  • To protect the healthcare worker if there is a break in his or her skin
  • To reduce risk of contact with infectious agents if gloves worn are punctured
  • To reduce the chances of disease transmission
• When healthcare personnel should wash hands

  • At the beginning and end of each shift
  • When the hands are visibly soiled
  • After contact with a possible source of microorganisms
  • Before and after performing invasive procedures
  • Before removing gloves if they are visibly soiled and each time after removing gloves
• Personal protective equipment (PPE)


  Specialized equipment and attire used in healthcare facilities to protect not only the healthcare workers but also the patients and visitors against infections