Chapter 40 Staining Specimens Microbiology Unit 7 Chapter 40 Staining Specimens Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Stains A variety of stains available Most common Gram stain Ziehl-Neelsen (acid-fast) stain Stain before culturing Rapid identification Determine appropriate medium Determine appropriate antibacterials Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Staining Kits Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Stains (cont.) Simple stains Crystal violet and methylene blue Typically used for yeasts Lactophenol cotton blue Confirms identity of fungal organisms Many stains available but most are performed only in large reference or research laboratories Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Gram Stain Used to categorize bacteria Requires four steps Gram-positive or gram-negative Based on cell wall structure Requires four steps Primary stain, a mordant, a decolorizer, and a counterstain Mordant – fixes dyes to the structures (cell wall) Primary stain usually crystal violet Mordant – Gram’s iodine solution Decolorizer – 95% ethanol or acetone Counterstain – basic fuchsin or safranin Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Gram Stain Procedure Thin sample applied to slide Swabs can be gently rolled on slide Sterile wire to one young colony (24 hour) Older colonies may not stain accurately Mix sample from plates with a drop of water or saline If broth – 2 to 3 loopfuls If liquid – circle drop area with a wax pencil No matter source – take care not to damage organisms Air dry then heat fix by passing through a flame 2 to 3 times, specimen side up Do not overheat – warm not hot Prevents sample from washing off, preserves morphology, kills the bacteria, and renders them permeable to the stain Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Gram Stain Procedure (cont.) Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Gram Stain Interpretation Gram-positive Bacteria that retain the violet-iodine complex stain purple Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Gram Stain Interpretation (cont.) Gram-negative Lose the crystal-violet or purple color and stain red Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Potassium Hydroxide Test Gram variable Stain both gram-positive and gram-negative May be due to excessive decolorization, an overly thick sample, excessive heat fixing, old cultures, or poor-quality stain KOH test Determines the true Gram status Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Potassium Hydroxide Test (cont.) A loopful (or two) of 3% HOH solution placed on slide A generous quantity of surface growth from culture transferred to the KOH Stir specimen (30 seconds to 2 minutes) Then gently lift the loop Gram-negative develop a mucoid appearance and produce a sticky strand Gram-positive – mixture remains homogeneous, no strand Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Ziehl-Neelsen Stain Primarily used to detect acid-fast organisms Mycobacterium and Nocardia species Many stains available, but few work well in veterinary practice laboratories Several steps Primary stain – dimethyl sulfoxide (DMSO) and carbol fuchsin Decolorizer – acid-alcohol Counterstain – methylene blue Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Ziehl-Neelsen Stain Procedure Slide is air dried and heat fixed Primary stain is flooded on slide Slide heated over flame until stain steams Cool slide for 5 minutes, then rinse with tap water Acid alcohol is used to decolorize – 1 to 2 minutes until red color is gone Rinse slide Counterstain added then rinsed with water and air dried Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Ziehl-Neelsen Stain (cont.) Agents like DMSO allow the stain to penetrate stain-resistant cells such as Mycobacterium If stain isn’t removed by acid alcohol then the organism is “acid-fast” and appears red Non–acid-fast cells stain blue Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Ziehl-Neelsen Stain (cont.) Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Giemsa Stain Used to detect spirochetes and rickettsiae and the capsule of Bacillus anthracis and the morphology of Dermatophilus congolensis Smear fixed with absolute methanol for 3 to 5 minutes and air dried Dip in diluted stain for 20 to 30 minutes For Borrelia anserina – the smear is gently heated while covered with the stain for 4 to 5 minutes then rinsed and air dried Purplish-blue-stained bacteria Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Specialized Stains Flagella, capsule, and endospore stains are available but have limited application in the average veterinary practice Expensive stains Malachite green endospore stain of Bacillus anthracis Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Summary Gram stain is the most common staining procedure in the microbiology laboratory Gram stains require primary stain, mordant, decolorizer, and counterstain Gram-positive – purple Gram-negative – red KOH test helps identify Gram variable organisms Ziehl-Neelsen stain is used to identify acid-fast organisms Flagella, capsule, and endospore stains are primarily used in reference laboratories Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.